Compositions and methods for inducing gene expression

ABSTRACT

The present invention provides recombinant nucleic acid molecules encoding a chimeric transactivator protein including a DNA binding domain of a DNA binding protein and a protein domain capable of transcriptional activation. The present invention also provides recombinant viral and non-viral vectors that are able to infect and/or transfect and sustain expression of a biologically active chimeric transactivator proteins in mammalian cells. Also provided are host cell lines and non-human transgenic animals capable of expressing biologically active chimeric transactivator proteins. In another aspect, compositions and methods for treating or preventing ischemic damage associated with hypoxia-related disorders are provided.

This application is a continuation of U.S. Ser. No. 09/579,897, filedMay 26, 2000, now issued as U.S. Pat. No. 6,432,927, which is acontinuation of PCT patent application PCT/US98/25753, filed Dec. 4,1998, which is a continuation-in-part of U.S. Ser. No. 09/133,612, filedAug. 13, 1998 now abandoned, which claims priority under 35 U.S.C. §119(e) to provisional application 60/067,546, filed Dec. 4, 1997.

BACKGROUND OF THE INVENTION

Ischemic heart disease occurs when the heart muscle does not receive anadequate blood supply and is thus deprived of necessary levels of oxygenand nutrients. Ischemia is commonly a result of atherosclerosis whichcauses blockages in the coronary arteries that provide blood flow to theheart muscle.

Ischemic heart disease can result in certain adaptive responses withinthe heart which are likely to be beneficial. Among these responsesare: 1) increased expression of angiogenic growth factors and theirreceptors, leading to the formation of collateral circulation aroundblocked coronary arteries; 2) increased expression of glycolytic enzymesas a means to activate a metabolic pathway which does not require O₂;and 3) expression of heat shock proteins which can protect the ischemictissue from death.

At least some of these responses appear to be regulated by a complexoxygen sensing mechanism which eventually leads to the activation oftranscription factors which control the expression of critical genesinvolved in this adaptation. Because this altered gene expression occursonly in response to hypoxia, which usually only occurs when a strainsuch as exercise is placed upon the diseased heart, cardiac patients donot usually receive much benefit from this endogenous compensatorymechanism. As a result, a number of conventional therapies attempt tosupplement the natural therapeutic responses of the heart to ischemia.

For example, such treatments include pharmacological therapies, coronaryartery bypass surgery and percutaneous revascularization usingtechniques such as balloon angioplasty. Standard pharmacological therapyis predicated on strategies that involve either increasing blood supplyto the heart muscle or decreasing the demand of the heart muscle foroxygen and nutrients.

Increased blood supply to the myocardium is achieved by agents such ascalcium channel blockers or nitroglycerin. These agents are thought toincrease the diameter of diseased arteries by causing relaxation of thesmooth muscle in the arterial walls. Decreased demand of the heartmuscle for oxygen and nutrients is accomplished either by agents thatdecrease the hemodynamic load on the heart, such as arterialvasodilators, or those that decrease the contractile response of theheart to a given hemodynamic load, such as beta-adrenergic receptorantagonists.

Surgical treatment of ischemic heart disease is based on the bypass ofdiseased arterial segments with strategically placed bypass grafts(usually saphenous vein or internal mammary artery grafts). Percutaneousrevascularization is based on the use of catheters to reduce thenarrowing in diseased coronary arteries. All of these strategies areused to decrease the number of, or to eradicate ischemic episodes, butall have various limitations.

More recently, delivery of angiogenic factors or heat shock proteins viaprotein or gene therapy has been proposed to further augment the heart'snatural response to ischemia. Indeed, various publications havediscussed the uses of gene transfer for the treatment or preventionheart disease. See, for example, Mazur et al., “Coronary Restenosis andGene Therapy”, Molecular and Cellular Pharmacology 21:104–111 (1994);French, B. A. “Gene Transfer and Cardiovascular Disorders” Herz.18(4):222–229 (1993); Williams, “Prospects for Gene Therapy of IschemicHeart Disease”, Am. J. Med. Sci. 306:129–136 (1993); Schneider andFrench “The Advent of Adenovirus: Gene Therapy for CardiovascularDisease” Circulation 88:1937–42 (1993). International Patent ApplicationNo. PCT/US93/11133, entitled “Adenovirus-Mediated Gene Transfer toCardiac and Vascular Smooth Muscle” reporting the use ofadenovirus-mediated gene transfer for regulating function in cardiacvascular smooth muscle.

Accordingly, there exists a need in the art for compositions and methodsfor inducing the expression of beneficial hypoxia-inducible genes inischemia-associated cells. Additionally, there exists a need for newvector compositions that allow efficient expression of a range ofpotentially beneficial genes that are activated by the sustained directexpression of a biologically active mammalian transcription factor. Thepresent invention satisfies these needs and provides related advantagesas well.

SUMMARY OF THE INVENTION

The present invention provides recombinant nucleic acid moleculesencoding a chimeric transactivator comprising a DNA binding domain of aDNA binding protein wherein the DNA binding protein is a mammalianhypoxia-inducible factor protein, and a functional transcriptionalactivator domain of a transcriptional activator protein.

Accordingly, in making the invention, we sought to exploit the adaptiveresponse to hypoxia as an alternative approach for the treatment ofischemia associated with vascular disease. We considered thatadministration of a modified HIF-1α transcription factor via genetherapy might induce expression of a panel of potentially beneficialgenes and ultimately lead to the neovascularization of ischemic tissues.We have created a constitutively active form of HIF-1α consisting of theDNA-binding and dimerization domains from HIF-1α and the transactivationdomain from herpes simplex virus VP16 protein. Among the possible targetgenes for this modified transcription factor is VEGF, an endothelialcell-specific mitogen and potent stimulator of angiogenesis.

In vitro analyses of an HIF-1α/VP16 hybrid transcription factor of theinvention demonstrated that activation of luciferase reporter constructsunder the transcriptional control of either the VEGF or EPO promoters aswell as up-regulation of endogenous VEGF gene expression in HeLa and C6cells was independent of induction. Experiments were performed in arabbit hindlimb ischemia model to test the hypothesis that exogenousadministration of a plasmid encoding HIF-1α/VP16 could enhancecollateral vessel formation and also to compare the potency ofHIF-1α/VP16 with that of VEGF as an angiogenic therapy. Results of thesestudies suggest that administration of DNA encoding a transcriptionfactor may represent a viable treatment strategy for tissue ischemia.

The present invention also provides recombinant viral and non-viralvectors that are able to infect and/or transfect and sustain expressionof a biologically active chimeric human-viral transactivator protein inmammalian cells.

The present invention further provides a recombinant plasmid vector(pcDNA3/HIF/VP16/Af12).

In another embodiment, the present invention provides a recombinantplasmid expression vector (pcDNA3/HIF/VP16/RI).

In yet another embodiment, the present invention provides mammaliancells and cell lines transfected with pcDNA3/HIF/VP16/Af12 orpcDNA3/HIF/VP16/RI.

In still yet another embodiment, the present invention providesrecombinant mammalian cell lines able to express biologically activechimeric human-viral transactivator protein at sustained levels.

The present invention also provides recombinant mammalian host celllines able to express and secrete biologically active chimerichuman-viral transactivator protein at sustained levels.

In another embodiment, the present invention provides a fusion proteincomprising a DNA binding domain of a DNA binding protein wherein the DNAbinding protein is the mammalian hypoxia-inducible factor 1α (HIF-1α)protein at the amino terminus, and a functional transcriptionalactivator domain of a transcriptional activator protein, wherein saidtranscriptional activator protein is HSV VP16 at the carboxy terminus.

The present invention further provides a non-human transgenic mammalexpressing recombinant DNA encoding a chimeric transactivator comprisinga DNA binding domain of a DNA binding protein wherein said DNA bindingprotein is the mammalian hypoxia-inducible factor 1α (HIF-1α) protein,and a functional transcriptional activator domain of a transcriptionalactivator protein, wherein said transcriptional activator protein is HSVVP16.

In yet another embodiment, the present invention provides a method forincreasing expression of hypoxia-inducible genes.

In still yet another embodiment, the present invention provides a methodfor providing sustained expression of biologically active HIF-1α undernormoxic conditions.

The present invention also provides a method fortreating/preventing/modulating hypoxia-associated tissue damage in asubject.

The present invention further provides a method for providingbiologically active chimeric human-viral transactivator protein to thecells of an individual comprising introducing into the cells of anindividual an amount of pcDNA3/HIF/VP16/RI or pcDNA3/HIF/VP16/Af12effective to transfect and sustain expression of biologically activechimeric human-viral transactivator protein in the cells of theindividual.

Other features and advantages of the present invention will be apparentfrom the following detailed description as well as from the claims.

All patent applications, patents, and literature references cited inthis specification are hereby incorporated by reference in theirentirety. In case of conflict or inconsistency, the present description,including definitions, will control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a restriction map of hybrid construct pcDNA3/HIF/VP16/Af12.

FIG. 2 shows a restriction map of hybrid construct pcDNA3/HIF/VP16/RI.

FIG. 3 shows the assay results testing the ability of the HIF-1α/VP16hybrid constructs to activate EPO or VEGF gene expression in 293 cells.

FIG. 4 shows the assay results testing the ability of the HIF-1α/VP16hybrid constructs to activate EPO or VEGF gene expression in HeLa cells.

FIG. 5 shows assay results testing the ability of the HIF-1α/VP16 hybridconstructs to activate expression of an endogenous VEGF gene in HeLacells.

FIG. 6 shows results testing the ability of the HIF-1α/VP16 hybridconstructs to activate EPO (left) or VEGF(right) gene expression in 293cells.

FIG. 7 shows a schematic of the adenoviral sequences contained with theHIF-1α/VP16 hybrid adenoviral vectors.

FIG. 8 shows assay results testing the ability of the HIF-1α/NFκBconstruct to activate expression of the endogenous VEGF gene in HeLacells (top) and the endogenous EPO gene in Hep3B cells (bottom).

DETAILED DESCRIPTION OF THE INVENTION

Hypoxia (a state in which O₂ demand exceeds supply) is a powerfulmodulator of gene expression. The physiologic response to hypoxiainvolves enhanced erythropoiesis (Jelkman, Physiol. Rev. 72:449–489(1992)), neovascularization in ischemic tissues (White et al., Circ.Res. 71:1490–1500 (1992)) and a switch to glycolysis-based metabolism(Wolfe et al., Eur. J. Biochem. 135:405–412 (1983)). These adaptiveresponses either increase O₂ delivery or activate alternate metabolicpathways that do not require O₂. The gene products involved in theseprocesses, include, for example: (i) EPO, encoding erythropoietin, theprimary regulator of erythropoiesis and thus a major determinant ofblood O₂-carrying capacity (Jiang et al., J. Biol. Chem.271(30):17771–78 (1996); (ii) VEGF, encoding vascular endothelial growthfactor, the primary regulator of angiogenesis and thus a majordeterminant of tissue perfusion (Levy et al., J. Biol. Chem. 270:13333(1995); Liu et al., Circ. Res. 77:638 (1995); Forsythe et al., Mol.Cell. Biol. 16:4604 (1996)); (iii) ALDA, ENO1, LDHA, PFKL, and PGK1,encoding the glycolytic enzymes aldolase A, enolase 1, lactatedehydrogenase A, phosphofructokinase L, and phosphoglycerate kinase 1,respectively, which provide a metabolic pathway for ATP generation inthe absence of O₂ (Firth et al., Proc. Natl. Acad. Sci., USA 91:6496(1994); Firth et al., J. Biol. Chem. 270:21021 (1995); Semenza et al.,J. Biol. Chem. 269:23757 (1994)); (iv) HO1 and iNOS, encoding hemeoxygenase 1 and inducible nitric oxide synthase, which are responsiblefor the synthesis of the vasoactive molecules carbon monoxide and nitricoxide, respectively (Lee et al., J. Biol. Chem. 272:5375; Melillo et al.J. Exp. Med. 182:1683 (1995)).

An important mediator of these responses is the interaction of atranscriptional complex comprising a DNA binding, hypoxia induciblefactor protein, with its cognate DNA recognition site, ahypoxia-responsive element (HRE) located within the promoter/enhancerelements of hypoxia-inducible genes. HREs consist of an hypoxiainducible factor protein binding site (that contains the core sequence5′-CGTG-3′) as well as additional DNA sequences that are required forfunction, which in some elements includes a second binding site.

HIF-1 is a heterodimeric protein composed of two subunits: (i) aconstitutively expressed beta (β) subunit (shared by other relatedtranscription factors) and (ii) an alpha (α) subunit (see, e.g., WO96/39426, International Application No. PCT/US96/10251 describing therecent affinity purification and molecular cloning of HIF-1α) whoseaccumulation is regulated by a post-translational mechanism such thathigh levels of the alpha subunit can only be detected during hypoxicconditions. Both subunits are members of the basic helix-loop-helix(bHLH)-PAS family of transcription factors. These domains regulate DNAbinding and dimerization. The transactivation domain is thought toreside in the C-terminus of the protein.

Whereas, HIF-1β (ARNT) is expressed constitutively at a high level,accumulation of HIF-1α in the cell is sensitive to O₂ concentration suchthat high levels are detected only during hypoxia. This observation hasled to a proposed mechanism for target gene activation whereby O₂concentration is detected by a sensor protein and through a complexsignaling mechanism leads to stabilization of the HIF-1α subunit. HIF-1αis then available to complex with HIF-1β and bind selectively to HREsites in the promoter/enhancer of the target gene(s). Regions of theHIF-1α protein involved in conferring this response are thought tocoincide with regions involved in transactivation.

Induction of HIF-1 activity in response to hypoxia is thought to occurvia stabilization of the HIF-1α protein. Regions of HIF-1α involved inthis response have been localized to the C-terminus of the protein andoverlap the transactivation domain. For example, Jiang et al., J. Biol.Chem. 271(30):17771–78 (1996) showed that HIF-1α truncated at amino acid390 lost transactivation activity but retained the ability to bind DNAand showed high levels of protein under both normoxic and hypoxicconditions. This result suggested that the transactivation domain aswell as the region conferring instability with normoxia reside in theC-terminal half of the protein. Pugh et al., J. Biol. Chem.272(17):11205–14 (1997) have further localized the regions involved totwo areas, amino acids 549–582 and 775–826.

In one embodiment, this invention provides nucleic acid moleculesencoding biologically active chimeric transactivator proteins comprisinga domain of the HIF-1α protein sufficient for DNA binding anddimerization with HIF-1β (ARNT) and a protein domain capable oftranscriptional activation.

In another embodiment, a related DNA binding, hypoxia inducible factorprotein is EPAS1. EPAS1 is a PAS domain transcription factor termedendothelial PAS-1. Tian et al., Genes Dev. 11:72 (1997). EPAS1 shares48% identity with HIF-1α and lesser similarity with other members ofbHLH/PAS domain family of transcription factors (EPAS1 human sequenceGenBank Acc. No. U81984; mouse sequence GenBank Ace. No. U81983). LikeHIF-1α, EPAS1 binds to and activates transcription from a DNA elementoriginally isolated from the EPO gene and containing the HRE coresequence. EPAS1 also forms a heterodimeric complex with ARNT prior totranscriptional activation of target genes.

Human and murine EPAS1 share extensive primary amino acid sequenceidentity with HIF-1α (48%). Sequence conservation between the twoproteins is highest in the bHLH (85%), PAS-A (68%), and PAS-B (73%)regions. A second region of sequence identity occurs at the extreme Ctermini of the EPAS1 and HIF-1α proteins. This conserved region inmHIF-1α has been shown to contain a hypoxia response domain (Li et al.,J. Biol. Chem. 271(35):21262–67 (1996)). The high degree of sequencesimilarity between EPAS1 and HIF-1α suggests that they share a commonphysiological function. Hypoxic conditions stimulate the ability ofHIF-1α to trans-activate a target gene containing the HRE core sequence.The activity of EPAS1 is also enhanced in cells grown under hypoxicconditions, suggesting that it may be subject to the same regulatoryinfluences as HIF-1α.

In yet another embodiment of the present invention the transcriptionfactor, DNA binding, hypoxia inducible factor protein is HLF. HLF is abHLH-PAS protein termed HIF-1α-like factor. Ema et al., Proc. Natl.Acad. Sci., USA 94:4273 (1997). HLF is a novel polypeptide of 874 aminoacids with a calculated molecular mass of 97 kDa (GenBank Accession No.D89787). Sequence comparison revealed that the amino acid sequence has astriking similarity to that of HIF-1α in the amino-terminal half(aa1–344) including bHLH (83.9%) and PAS (66.5%) motifs followed by asequence with a moderate similarity (36.4%, aa 345–559). While most ofthe sequence in the C-terminal half was variable between this proteinand HIF-1α, a small portion (63%, aa 824–874) of noticeable sequencesimilarity was found in the very C terminus. That basic amino acids ofthe bHLH region involved in DNA recognition are completely conservedamong HLF and HIF-1α, suggests that these factors recognize verysimilar, if not identical, regulatory DNA sequences. Experiments showedthat both HLF and HIF-1α bound the VEGF and EPO HRE sequences inassociation with ARNT with a similar affinity.

In another embodiment, the chimeric transactivator proteins of thisinvention comprise a domain of a non-mammalian hypoxia inducible factorprotein. As will be recognized by the skilled artisan, the adaptiveresponse to hypoxia is likely to have been highly conserved throughoutevolution. Accordingly, hypoxia inducible factor proteins would beexpected to occur in a wide variety of species including non-mammalianvertebrates and non-vertebrates such as insects. See, for example, Baconet al., Biochem. Biophys. Res. Comm., 249:811–816 (1998), which reportsthe functional similarity between the Sima basic-helix-loop-helix PASprotein from Drosophila and the mammalian HIF-1α protein.

Nucleic acid and amino acid sequences for non-mammalian hypoxiainducible factor proteins may be obtained by the skilled artisan by avariety of techniques, for example by cross-hybridization oramplification using all or a portion of the sequences referred toherein. Once the sequence encoding a candidate hypoxia inducible factorprotein has been determined, the localization of portions of the proteinsufficient to bind to HREs and dimerize with HIF-1β may be determinedusing, e.g., the same types of techniques used to determine the locationof those domains within the human HIF-1α protein. Relevant domains ofnon-mammalian hypoxia inducible factor proteins useful in thecompositions and methods of this invention may also be producedsynthetically or by site-directed manipulations of the DNA encodingknown mammalian hypoxia inducible factor proteins. It is also expectedthat the sequence motifs in common among various mammalian andnon-mammalian hypoxia inducible factor proteins will suggest consensussequences that, while perhaps not occurring naturally in any species,would nevertheless produce domains useful in the methods andcompositions of this invention. All that is required in order tosubstitute such non-mammalian hypoxia inducible factor protein domainsfor the human HIF-1α protein domains exemplified herein is that they beable to bind to HREs and dimerize with HIF-1β (ARNT).

Accordingly, it is proposed to modify the hypoxia inducible factorprotein by removing the C-terminal (transactivation)domain and replacingit with a strong transactivator sequence. This modification should notalter its ability to dimerize with the β/ARNT subunit or bind tospecific DNA sequences (e.g., HREs) but could convert the hypoxiainducible factor protein into a constitutive inducer of potentiallytherapeutic genes (for example, VEGF, EPO, phosphoglycerate kinase, andthe like).

Replacement of the C terminal (or transactivation) region of the hypoxiainducible factor protein with a strong transactivation domain from atranscriptional activator protein such as, for example, Herpes SimplexVirus (HSV) VP16 or yeast transcription factors GAL4 and GCN4, isdesigned to stabilize the protein under normoxic conditions and providestrong, constitutive, transcriptional activation. Administration of thisprotein to the cells of a subject via gene therapy should be aneffective treatment or prophylactic for chronic ischemia due to coronaryartery disease, peripheral vascular disease as well as ischemic diseaseof the limb.

In the present application, of interest is the ability of hypoxiainducible factor proteins, for example, HIF-1α, EPAS1 and HLF to induceexpression of hypoxia-inducible genes such as, for example VEGF and thelike, resulting in the amelioration of symptoms through promotion ofcollateral blood vessel growth.

For example, although the HIF-1α subunit is unstable during normoxicconditions, overexpression of this subunit in cultured cells undernormal oxygen levels is capable of inducing expression of genes normallyinduced by hypoxia. This suggests that a useful gene therapy strategymight be to express high levels of the HIF-1α subunit in ischemic heartin vivo using a recombinant plasmid or viral vector. An alternativestrategy would be to modify the HIF-1α subunit such that it no longer isdestabilized by normoxic conditions and would therefore be more potent,particularly when the patient being treated is not actually ischemic.

To stabilize the hypoxia inducible factor protein under normoxicconditions and to provide strong, constitutive transcriptionalactivation, a hybrid/chimeric fusion protein consisting of theDNA-binding and dimerization domains from HIF-1α and the transactivationdomain from Herpes Simplex Virus (HSV) VP16 protein was constructed.Administration of this hybrid/chimera to the cells of a subject via genetherapy will theoretically induce the expression of genes normallyup-regulated in response to hypoxia (i.e., VEGF and the like).

Alternative biologically active chimeric transactivator proteins, suchas a protein comprising the DNA binding and dimerization domain fromHIF-1α and the transactivation domain from the human NFκB protein, areexpected to produce similar results.

“Hypoxia” means the state in which O₂ demand exceeds supply.

“Hypoxia-inducible genes” means genes containing one or more hypoxiaresponsive elements (HREs; binding sites) within sequences mediatingtranscriptional activation in hypoxic cells.

Hypoxia inducible factor means a DNA binding protein/transcriptionfactor the expression of which is upregulated under hypoxic conditions,that recognizes and binds to a hypoxia responsive element core sequencewithin a gene and thereby activates such gene.

Hypoxia-associated disorders include, for example, ischemic heartdisease, peripheral vascular disease, ischemic disease of the limb, andthe like.

The term “nucleic acids” (also referred to as polynucleotides)encompasses RNA as well as single and double-stranded DNA, cDNA andoligonucleotides.

Nucleic acids also encompass isolated nucleic acid sequences, includingsense and antisense oligonucleotide sequences, e.g., derived from theHIF-1α, EPAS1, or the HLF sequences. HIF-1α, EPAS1-, or HLF-derivedsequences may also be associated with heterologous sequences, includingpromoters, enhancers, response elements, signal sequences,polyadenylation sequences, and the like. As used herein, the phrase“isolated” means a polynucleotide that is in a form that does not occurin nature. One means of isolating polynucleotides is to probe a humantissue-specific library with a natural or artificially designed DNAprobe using methods well known in the art. DNA probes derived from thehuman HIF-1α gene, EPAS1, or the HLF gene are particularly useful forthis purpose. DNA and cDNA molecules that encode invention polypeptidescan be used to obtain complementary genomic DNA, cDNA or RNA from human,mammalian, or other animal sources, or to isolate related cDNA orgenomic clones by the screening of cDNA or genomic libraries, by methodsdescribed in more detail below.

Furthermore, the nucleic acids can be modified to alter stability,solubility, binding affinity, and specificity. For example,invention-derived sequences can further include nuclease-resistantphosphorothioate, phosphoroamidate, and methylphosphonate derivatives,as well as “protein nucleic acid” (PNA) formed by conjugating bases toan amino acid backbone as described in Nielsen et al., Science,254:1497, (1991). The nucleic acid may be derivatized by linkage of theα-anomer nucleotide, or by formation of a methyl or ethylphosphotriester or an alkyl phosphoramidate linkage. Furthermore, thenucleic acid sequences of the present invention may also be modifiedwith a label capable of providing a detectable signal, either directlyor indirectly. Exemplary labels include radioisotopes, fluorescentmolecules, biotin, and the like.

In general, nucleic acid manipulations according to the presentinvention use methods that are well known in the art, as disclosed in,for example, Sambrook et al., Molecular Cloning, A Laboratory Manual 2dEd. (Cold Spring Harbor, N.Y., 1989), or Ausubel et al., CurrentProtocols in Molecular Biology (Greene Assoc., Wiley Interscience, NY,N.Y., 1992).

This invention also encompasses nucleic acids which differ from thenucleic acids encoding a human HIF-1α, EPAS1, or HLF, but which have thesame phenotype, i.e., encode substantially the same amino acid sequence,respectively. Phenotypically similar nucleic acids are also referred toas “functionally equivalent nucleic acids”. As used herein, the phrase“functionally equivalent nucleic acids” encompasses nucleic acidscharacterized by slight and non-consequential sequence variations thatwill function in substantially the same manner to produce the same orsubstantially the same protein product(s) as the nucleic acids disclosedherein. In particular, functionally equivalent nucleic acids encodeproteins that are the same as those disclosed herein or that haveconservative amino acid variations. For example, conservative variationsinclude substitution of a non-polar residue with another non-polarresidue, or substitution of a charged residue with a similarly chargedresidue. These variations include those recognized by skilled artisansas those that do not substantially alter the tertiary structure of theprotein.

A structural gene is that portion of a gene comprising a DNA segmentencoding a protein, polypeptide or a portion thereof, and excluding the5′ sequence which drives the initiation of transcription. The structuralgene may be one which is normally found in the cell or one which is notnormally found in the cellular location wherein it is introduced, inwhich case it is termed a heterologous gene. A heterologous gene may bederived in whole or in part from any source know to the art, including abacterial genome or episome, eukaryotic, nuclear or plasmid DNA, cDNA,vital DNA or chemically synthesized DNA. A structural gene may containone or more modifications in either the coding or the untranslatedregions which could affect the biological activity or the chemicalstructure of the expression product, the rate of expression or themanner of expression control. Such modifications include, but are notlimited to, mutations, insertions, deletions and substitutions of one ormore nucleotides. The structural gene may constitute an uninterruptedcoding sequence or it may include one or more introns, bound by theappropriate splice junctions. The structural gene may be a composite ofsegments derived from a plurality of sources, naturally occurring orsynthetic. The structural gene may also encode a fusion protein. It iscontemplated that the introduction of recombinant DNA moleculescontaining the structural gene/transactivator complex will includeconstructions wherein the structural gene and the transactivator areeach derived from different sources or species.

Eukaryotic transcription factors are often composed of separate andindependent DNA binding and transcriptional activator domains (Mitchelland Tjian, Science 245:371–378 (1989)). The independence of the domainshas allowed for the creation of functional fusion proteins consisting ofthe DNA binding and activating domains of heterologous proteins.Chimeric eukaryotic regulatory proteins, consisting of the lexA DNAbinding protein and the activation domain of the yeast transcriptionfactor, GAL4, were constructed by Brent and Ptashne (Nature 312:612–615(1985)). The use of fusion proteins has identified several types ofprotein domains which act as transcriptional activators. These domainshave little amino acid similarity but often are characterized as beingeither highly acidic (as in the case of GAL4 and GNC4), glutamine-rich(as in the case of Sp1), or proline-rich (as in the case of NF1, Ma andPtashne, Cell 51:113–119 (1987); Courey and Tjian (1988); Mermod et al.,Cell 58:741–753 (1989)).

One of the most efficient activator domains known is contained in thecarboxyl-terminal 100 amino acids of the Herpes Simplex Virus (HSV)virion protein 16 (VP16; Sadowski et al., Nature 335:563–564 (1988);Triezenberg et al., Genes & Dev. 2:718–729 (1988)). VP16, also known asVmw65 or alpha-gene trans-inducing factor, is a structural protein ofHSV which activates transcription of the immediate early promoters ofthe virus, including those for ICPO and ICP4 (Campbell et al., J. Mol.Biol. 180:1–19 (1984); Kristie and Roizman, Proc. Natl. Acad. Sci., USA81:4065–4069 (1984); Pellet et al., Proc. Natl. Acad. Sci., USA82:5870–5874 (1985)). Although VP16 specifically activates promoterscontaining the so called TAATGARAT element, the specificity is endowedby a cellular DNA binding protein(s) which is complexed with the aminoterminal domains(s) of VP16 (McKnight et al., Proc. Natl. Acad. Sci.,USA 84:7061–7065 (1987); Preston et al., Cell 52:425–434 (1988)).

The present invention provides novel hybrid/chimeric transactivatingproteins comprising a functional portion of a DNA binding protein and afunctional portion of a transcriptional activator protein. Thehybric/chimeric transactivating proteins of the invention offer avariety of advantages, including the specific activation of expressionof hypoxia-inducible genes containing hypoxia responsive elements(HREs), thereby achieving exceptionally high levels of gene expression.Invention hybrid/chimeric transactivating proteins are capable offunctioning in vertebrate cells and may include naturally occurringtranscriptional transactivating proteins or domains of proteins fromeukaryotic cells including vertebrate cells, viral transactivatingproteins or any synthetic amino acid sequence that is able to stimulatetranscription from a vertebrate promoter. Examples of suchtransactivating proteins include, but are not limited to, the lymphoidspecific transcription factor identified by Muller et al. (Nature336:544–551 (1988)), the fos protein (Lucibello et al., Oncogene 3:43–52(1988)); v-jun protein (Bos et al., Cell 52:705–712 (1988)); factor EF-C(Ostapchuk et al., Mol. Cell. Biol. 9:2787–2797 (1989)); HIV-1 tatprotein (Arya et al., Science 229:69–73 (1985)), the papillomavirus E2protein (Lambert et al., J. Virol. 63:3151–3154 (1989)) the adenovirusE1A protein (reviewed in Flint and Shenk, Ann. Rev. Genet. (1989), heatshock factors (HSF1 and HSF2) (Rabindran, et al., PNAS 88:6906–6910(1991)); the p53 protein (Levine, Cell 88:323–331 (1997), Ko and Prives,Genes Dev. 10:1054–1072 (1996)); Sp1 (Kadonaga, et al. Cell 51:1079–1090(1987)); AP1 (Lee, et al., Nature 325:368–372 (1987)); CTF/NF1 (Mermod,et al., Cell 58: 741–753 (1989)), E2F1 (Neuman, et al., Gene 173:163–169 (1996)); HAP1 (Pfeifer, et al., Cell 56: 291–301 (1989)); HAP2(Pinkham, et al., Mol. Cell. Biol. 7:578–585 (1987)); MCM1 (Passmore, etal., J. Mol. Biol. 204:593–606 (1988); PHO2 (Sengstag, and Hinnen, NAR15:233–246 (1987)); and GAL11 (Suzuki et al., Mol. Cell. Biol.8:4991–4999 (1988)). In preferred embodiments of the invention, thetransactivating protein is Herpes simplex virus VP16 (Sadowski et al.,Nature 335:563–564 (1988); Triezenberg et al., Genes and Dev. 2:718–729(1988)), NFκB ((Schmitz and Baeuerle, EMBO J. 10:3805–3817 (1991);Schmitz, et al., J. Biol. Chem. 269:25613–25620 (1994); and Schmitz, etal., J. Biol. Chem. 270:15576–15584 (1995)), and yeast activators GAL4and GCN4.

Of course, the skilled artisan will understand that transcriptionalactivation domains useful in the compositions and methods of thisinvention may also be synthetic, i.e., based on a sequence that is notcontained within a known, naturally occurring protein. See, for example,Pollock and Gilman, PNAS 94:13388–13389 (1997), which teaches thattranscriptional activation is an inherently flexible process in whichthere is little, if any, requirement for specific structures orstereospecific protein contacts. It also reviews the variety ofdifferent molecules that can function as transcriptional activators,including short peptide motifs (as small as eight amino acids), simpleamphipathic helices and even mutagenized domains of proteins unrelatedto transcriptional activation.

According to the invention, DNA sequences encoding the DNA bindingprotein and the transactivating protein are combined so as to preservethe respective binding and transactivating properties of each. Invarious embodiments of the invention, the DNA encoding thetransactivating protein, or a portion thereof capable of activatingtranscription, may be inserted into DNA at a locus which does notcompletely disrupt the function of said DNA binding protein. Regions notrequired for function of DNA binding proteins or transcriptionaltransactivating proteins may be identified by any method known in theart, including analysis of mapped mutations as well as identification ofregions lacking mapped mutations, which are presumably less sensitive tomutation than other, more functionally relevant portions of themolecule. The appropriate recombinant constructs may be produced usingstandard techniques in molecular biology, including those set forth inManiatis (Molecular Cloning: A Laboratory Manual (Cold Spring Harbor,N.Y., Cold Spring Harbor Laboratory (1989)).

The recombinant DNA construct encoding the chimeric transactivatorprotein may be placed under the control of (i.e., operatively linked to)a suitable promoter and/or other expression control sequence. It may bedesirable for the transactivator protein to be placed under the controlof a constitutively active promoter sequence, although saidtransactivator protein may also be placed under the control of aninducible promoter, such as the metallothionine promoter (Brinster etal., Nature 296:39–42 (1982)) or a tissue specific promoter. Promotersequences which may be used according to the invention include, but arenot limited to, the SV40 early promoter region (Benoist and Chambon,Nature 290:304–310 (1981)), the promoter contained in the long terminalrepeat of Rous sarcoma virus (Yamamoto, et al., Cell 22:787–797 (1980)),the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad.Sci., U.S.A. 78:144–1445 (1981)), the human cytomegalovirus (CMV)immediate early promoter/enhancer (Boshart et al., Cell 41:521–530(1985)), and the following animal transcriptional control regions, whichexhibit tissue specificity and have been utilized in transgenic animals:elastase I gene control region which is active in pancreatic acinarcells (Swift et al., Cell 38:639–646 (1984); Ornitz et al., Cold SpringHarbor Symp. Quant. Biol. 50:399–409 (1986); MacDonald, Hepatology7:425–515 (1987)); insulin gene control region which is active inpancreatic beta cells (Hanahan, Nature 315:115–122 (1985)),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., Cell 38:647–658 (1984); Adames et al., Nature318:533–538 (1985); Alexander et al., Mol. Cell. Biol. 7:1436–1444(1987)), mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., Cell45:485–495 (1986)), albumin gene control region which is active in liver(Pinkert et al., Genes and Devel. 1:268–276 (1987)), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., Mol.Cell. Biol. 5:1639–1648 (1985); Hammer et al., Science 235:53–58(1987)); alpha 1-antitrypsin gene control region which is active in theliver (Kelsey et al, Genes and Devel. 1:161–171 (1987)), beta-globingene control region which is active in erythroid cells (Mogram et al.,Nature 315:338–340 (1985); Kollias et al., Cell 46:89–94 (1986)); myelinbasic protein gene control region which is active in oligodendrocytecells in the brain (Readhead et al., Cell 48:703–712 (1987)); myosinlight chain-2 gene control region which is active in skeletal muscle(Sani, Nature 314:283–286 (1985)), and gonadotropic releasing hormonegene control region which is active in the hypothalamus (Mason et al.,Science 234:1372–1378 (1986)). Of particular interest is the α-myosinheavy chain gene (Subramaniam, et al., J. Biol. Chem. 266:24613–24620,(1991)) and the myosin light chain-2 promoter (Henderson et al., J.Biol. Chem. 264:18142–18148 (1989) and Ruoqian-Shen et al., Mol. Cell.Biol. 11:1676–1685 (1991), both of which are active in cardiac muscle.

In one preferred specific embodiment of the invention, the chimerictransactivator protein is encoded by pcDNA3/HIF/VP16/Af12, constructedaccording to methods set forth in Example 1 and FIG. 1. In anotherpreferred specific embodiment of the invention, the chimerictransactivator protein is encoded by pcDNA3/HIF/VP16/RI, which isidentical to pcDNA3/HIF/VP16/Af12 except that the VP16 segment isinserted after codon 530 of the HIF-1α coding region.

According to the invention, the hybrid/chimeric transactivator proteinsof the invention may be utilized to specifically regulate the expressionof genes containing hypoxia responsive elements (HREs). These HREscorrespond to a nucleic acid sequence recognized and bound by the DNAbinding protein used as the backbone of the chimeric transactivatorprotein.

In general, the chimeric transactivator proteins of the invention may beused to selectively control the expression of genes of interest. Forexample, and not by way of limitation, the chimeric transactivatorproteins of the invention may be placed under control of a constitutivepromoter and may be used to constitutively increase the expression of agene of interest associated with hypoxia responsive elements (HREs), forexample, when it is desirable to produce a particular gene product inquantity in a cell culture or in a transgenic animal. Alternatively, thetransactivator protein may be placed under the control of atissue-specific promoter so that the gene of interest is expressed in aparticular tissue. In alternative embodiments of the invention, thechimeric transactivator function is inducible, so that the expression ofa gene of interest, via hypoxia responsive elements (HREs), may beselectively increased or decreased. For reviews of conditional andinducible transgene expression, see Fishman, Circ. Res., 82:837–844(1998) and Fishman, Trends Cardiovasc. Med., 5:211–217 (1995).

The chimeric transactivating proteins possess the advantageous propertyof binding specifically to responsive elements homologous to DNAsequences recognized by the chimeric protein's DNA binding proteinbackbone.

Vectors: Examples of vectors are viruses, such as adenoviruses,adeno-associated viruses (AAV), lentiviruses, herpes viruses, positivestrand RNA viruses, vaccinia viruses, baculoviruses and retroviruses,bacteriophages, cosmids, plasmids, fungal vectors and otherrecombination vehicles typically used in the art which have beendescribed for expression in a variety of eukaryotic and prokaryotichosts, and may be used for gene therapy as well as for simple proteinexpression.

Polynucleotides/transgenes are inserted into vector genomes usingmethods well known in the art. For example, insert and vector DNA can becontacted, under suitable conditions, with a restriction enzyme tocreate complementary ends on each molecule that can pair with each otherand be joined together with a ligase. Alternatively, synthetic nucleicacid linkers can be ligated to the termini of restricted polynucleotide.These synthetic linkers contain nucleic acid sequences that correspondto a particular restriction site in the vector DNA. Additionally, anoligonucleotide containing a termination codon and an appropriaterestriction site can be ligated for insertion into a vector containing,for example, some or all of the following: a selectable marker gene,such as the neomycin gene for selection of stable or transienttransfectants in mammalian cells; enhancer/promoter sequences from theimmediate early gene of human CMV for high levels of transcription;transcription termination and RNA processing signals from SV40 for mRNAstability; SV40 polyoma origins of replication and ColE1 for properepisomal replication; versatile multiple cloning sites; and T7 and SP6RNA promoters for in vitro transcription of sense and antisense RNA.Other means are well known and available in the art.

The skilled artisan will recognize that when expression from the vectoris desired, the polynucleotides/transgenes are operatively linked toexpression control sequences. Vectors that contain both a promoter and acloning site into which a polynucleotide can be operatively linked arewell known in the art. Such vectors are capable of transcribing RNA invitro or in vivo, and are commercially available from sources such asStratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.). Inorder to optimize expression and/or in vitro transcription, it may benecessary to remove, add or alter 5′ and/or 3′ untranslated portions ofthe clones to eliminate extra, potential inappropriate alternativetranslation initiation codons or other sequences that may interfere withor reduce expression, either at the level of transcription ortranslation. Alternatively, consensus ribosome binding sites can beinserted immediately 5′ of the start codon to enhance expression.Similarly, alternative codons, encoding the same amino acid, can besubstituted for coding sequences of the human HIF-1α, EPAS1 or HLFpolypeptide in order to enhance transcription (e.g., the codonpreference of the host cell can be adopted, the presence of G-C richdomains can be reduced, and the like).

Preparations of invention polynucleotides encoding human HIF-1α, EPAS1and HLF or another hypoxia inducible factor protein can be incorporatedin a suitable vector for delivery into a individual's cells usingmethods that are known in the art. See, for example, Finkel and Epstein,FASEB J. 9:843–851 (1995); Feldman et al., Cardiovascular Res.32:194–207 (1996).

In one embodiment, this invention provides compositions comprising apharmaceutically acceptable carrier and nucleic acid molecules capableof expressing biologically active chimeric transactivator proteins. Thechimeric transactivator proteins encoded by the nucleic acid moleculesinclude a DNA binding domain from a hypoxia inducible factor protein anda protein domain capable of transcriptional activation. Such domain maybe from either a naturally occurring or synthetic transcriptionalactivator molecule. The nucleic acid molecules within the compositionare in a form suitable for delivery into cells in vivo or in vitro. Avariety of such forms are well known in the art. Given the teachings setforth herein, the skilled artisan may select among various vectors andother expression/delivery elements depending on such factors as the siteand route of administration and the desired level and duration ofexpression.

Naked DNA—Naked plasmid DNA can be introduced into muscle cells, forexample, by direct injection into the tissue. (Wolff et al., Science247:1465 (1989)).

DNA-Lipid Complexes—Lipid carriers can be associated with naked DNA(e.g., plasmid DNA) to facilitate passage through cellular membranes.Cationic, anionic, or neutral lipids can be used for this purpose.However, cationic lipids are preferred because they have been shown toassociate better with DNA which, generally, has a negative charge.Cationic lipids have also been shown to mediate intracellular deliveryof plasmid DNA (Felgner and Ringold, Nature 337:387 (1989)). Intravenousinjection of cationic lipid-plasmid complexes into mice has been shownto result in expression of the DNA in lung (Brigham et al., Am. J. Med.Sci. 298:278 (1989)). See also, Osaka et al., J. Pharm. Sci.85(6):612–618 (1996); San et al., Human Gene Therapy 4:781–788 (1993);Senior et al., Biochemica et Biophysica Acta 1070:173–179 (1991);Kabanov and Kabanov, Bioconjugate Chem. 6:7–20 (1995); Remy et al.,Bioconjugate Chem. 5:647–654 (1994); Behr, J-P., Bioconjugate Chem.5:382–389 (1994); Behr et al., Proc. Natl. Acad. Sci., USA 86:6982–6986(1989); and Wyman et al., Biochem. 36:3008–3017 (1997).

Cationic lipids are known to those of ordinary skill in the art.Representative cationic lipids include those disclosed, for example, inU.S. Pat. No. 5,283,185; and PCT/US95/16174 (WO 96/18372), thedisclosures of which are incorporated herein by reference. In apreferred embodiment, the cationic lipid is N⁴-spermine cholesterolcarbamate (GL-67) disclosed in WO 96/18372.

Adenovirus—Adenovirus-based vectors for the delivery of transgenes arewell known in the art and may be obtained commercially or constructed bystandard molecular biological methods. Recombinant adenoviral vectorscontaining exogenous genes for transfer are, generally, derived fromadenovirus type 2 (Ad2) and adenovirus type 5 (Ad5). They may also bederived from other non-oncogenic scrotypes. See, for example, Horowitz,“Adenoviridae and their Replication” in VIROLOGY, 2d ed., Fields et al.Eds., Raven Press Ltd., New York, 1990, incorporated herein byreference.

The adenoviral vectors of the present invention are incapable ofreplicating, have minimal viral gene expression and are capable ofexpressing a transgene in target cells. Adenoviral vectors are generallyrendered replication-defective by deletion of the E1 region genes. Thereplication-defective vectors maybe produced in the 293 cell line (ATCCCRL 1573), a human embryonic kidney cell line expressing E1 functions.The deleted E1 region may be replaced by the transgene of interest underthe control of an adenoviral or non-adenoviral promoter. The transgenemay also be placed in other regions of the adenovirus genome. See,Graham et al., “Adenovirus-based Expression Vectors and RecombinantVaccines” in VACCINES: NEW APPROACHES to IMMUNOLOGICAL PROBLEMS pp363–390, Ellis, Ed., Butterworth-Heinemann, Boston, (1992) for a reviewof the production of replication-defective adenoviral vectors, alsoincorporated herein by reference.

Skilled artisans are also aware that other non-essential regions of theadenovirus can be deleted or repositioned within the viral genome toprovide an adenoviral vector suitable for delivery of a transgene inaccordance with the present invention. For example, PCT/US93/11667 (WO94/12649) and U.S. Pat. No. 5,670,488, incorporated herein by reference,discloses that some or all of the E1 and E3 regions may be deleted, andnon-essential open reading frames (ORFs) of E4 can also be deleted.Other representative adenoviral vectors are disclosed, for example, byRich et al., Human Gene Therapy 4:461 (1993); Brody et al., Ann. NYAcad. Sci. 716:90 (1994); Wilson, N. Eng. J. Med. 334:1185 (1996);Crystal, Science 270:404 (1995); O'Neal et al., Hum. Mol. Genet. 3:1497(1994); and Graham et al., supra., incorporated herein by reference. Ina preferred embodiment of the present invention, the adenoviral vectoris an E1 deleted Ad2-based vector.

In the adenoviral vectors of the present invention, thepolynucleotide/transgene is operably linked to expression controlsequences, e.g., a promoter that directs expression of the transgene. Asused herein, the phrase “operatively linked” refers to the functionalrelationship of a polynucleotide/transgene with regulatory and effectorsequences of nucleotides, such as promoters, enhancers, transcriptionaland translational stop sites, and other signal sequences. For example,operative linkage of a polynucleotide to a promoter refers to thephysical and functional relationship between the polynucleotide and thepromoter such that transcription of DNA is initiated from the promoterby an RNA polymerase that specifically recognizes and binds to thepromoter, and wherein the promoter directs the transcription of RNA fromthe polynucleotide.

Promoter regions include specific sequences that are sufficient for RNApolymerase recognition, binding and transcription initiation.Additionally, promoter regions include sequences that modulate therecognition, binding and transcription initiation activity of RNApolymerase. Such sequences may be cis acting or may be responsive totrans acting factors. Depending upon the nature of the regulation,promoters may be constitutive or regulated. Examples of promoters areSP6, T4, T7, SV40 early promoter, cytomegalovirus (CMV) promoter, mousemammary tumor virus (MMTV) steroid-inducible promoter, Moloney murineleukemia virus (MMLV) promoter, phosphoglycerate kinase (PGK) promoter,and the like. Alternatively, the promoter may be an endogenousadenovirus promoter, for example the E1a promoter or the Ad2 major latepromoter (MLP). Similarly, those of ordinary skill in the art canconstruct adenoviral vectors utilizing endogenous or heterologous poly Aaddition signals.

As used herein “promoter” refers to the nucleotide sequences at the 5′end of a structural gene which direct the initiation of transcription.Promoter sequences are necessary, but not always sufficient, to drivethe expression of a downstream gene. In general, eukaryotic promotersinclude a characteristic DNA sequence homologous to the consensus 5′TATA box about 10–30 bp 5′ to the transcription start site (CAP site).Another promoter component, the CAAT box, is often found about 30–70 bp5′ to the TATA box.

As used herein “enhancer” refers to a eukaryotic promoter sequenceelement that appears to increase transcriptional efficiency in a mannerrelatively independent of position and orientation with respect to anearby gene (Khoury and Gruss (1983) Cell 33:313–314). The ability ofenhancer sequences to function upstream from, within or downstream fromeukaryotic genes distinguishes them from classic promoter elements.

The viral and non-viral vectors of the present invention are useful fortransferring a polynucleotide/transgene to a target cell. The targetcell may be in vitro or in vivo. Use of invention vectors in vitroallows the transfer of a polynucleotide/transgene to a cultured cell andis useful for the recombinant production of the polynucleotide/transgeneproduct. In vitro methods are also useful in ex vivo gene therapymethods, in which a transgene is introduced into cells in vitro and thecells are then implanted into an individual. The skilled artisan willrecognize that in employing such techniques, the transgene may beintroduced into freshly isolated cells or cultured cells. Furthermore,the transgene-containing cells may be implanted immediately afterintroduction of the transgene or may be cultured prior to implantation.

The vectors of this invention find use in a variety of ex vivo genetherapy methods useful for prevention or treatment of ischemia and otherhypoxia-associated disorders. For example, it has been reported thattransplantation of cultured cardiomyocytes into myocardial scar tissuemay prevent heart failure (Ren-Ke Li et al., Ann. Thorac. Surg.62:654–661 (1996). It has also been reported that various combinationsof growth factors are capable of inducing cardiogenesis in cells ofnon-cardiac lineages (see PCT/US97/14229). Given the teachings containedherein, the skilled artisan will understand that introduction of anucleic acid molecule capable of expressing a chimeric transactivatorprotein according to this invention into target cells prior toimplantation in vivo may provide additional advantages to cellulartherapy methods in at least two ways. First, the cells may serve as atransport vehicle for the expression construct, resulting insite-directed delivery of the chimeric transactivator protein in anyregion of the body in which the cells are transplanted. Second, theexpression of a chimeric transactivator protein in the implanted cellsmay aid their survival after implantation, either by allowing them tomore easily adapt to any hypoxic conditions which may be present afterimplant, and/or by stimulating blood vessel development in the region ofimplantation.

Use of invention vectors to deliver a polynucleotide/transgene to a cellin vivo is useful for the treatment of various disorders, for example,in the case of hypoxia-associated disorders such as ischemic heartdisease, to a cell in which HIF-1α is absent, insufficient ornonfunctional. Thus, in further embodiments, this invention providesmethods for increasing the expression of hypoxia-inducible genes intarget cells of a subject in which such increased expression is desiredby administering an effective amount of a composition comprising nucleicacid molecule encoding a biologically active chimeric transactivatorprotein according to this invention in form suitable for expression(e.g., operatively linked to expression control sequences). An“effective amount” refers to an amount which results in expression ofbiologically active chimeric transactivator protein at a level and for aperiod of time sufficient to alleviate one or more of the symptomsassociated with a hypoxia-associated disorder. Such methods are usefulto increase or sustain the expression of HIF-1α and hypoxia-induciblegenes in tissues under hypoxic and normoxic conditions.

In related embodiments, the invention provides methods for treating,reducing and/or preventing hypoxia-associated tissue damage in a subjectby in vivo or ex vivo administration of an effective amount of thenucleic acid molecules of this invention to cells in which expression ofa biologically active chimeric transactivator protein is desired. Thetreatment and/or prevention of hypoxia-associated tissue damage may bemanifested by a variety of physiological effects, including increasedperfusion into a previously ischemic region of tissue. The methods ofthis invention find use in the treatment of such disorders as coronaryartery disease and peripheral vascular disease, including critical limbischemia.

In vivo administration of the compositions of this invention may beeffected by a variety of routes including intramuscular, intravenous,intranasal, subctaneous, intubation, lavage and intra-arterial delivery.Such methods are well known to the skilled artisan. Likewise, theprecise effective amount of the composition to be administered may bedetermined by the skilled artisan with consideration of factors such asthe specific components of the composition to be administered, the routeof administration, and the age, weight, extent of disease and physicalcondition of the subject being treated.

Also provided by this invention are vectors comprising a polynucleotideencoding human HIF-1α, EPAS1, HLF polypeptide and domains of otherhypoxia, inducible factor proteins, adapted for expression in abacterial cell, a yeast cell, an amphibian cell, an insect cell, amammalian cell and other animal cells. The vectors additionally comprisethe regulatory elements necessary for expression of the polynucleotidein the bacterial, yeast, amphibian, mammalian or animal cells so locatedrelative to the polynucleotide as to permit expression thereof.

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA, if an appropriateeukaryotic host is selected. Regulatory elements required for expressioninclude promoter sequences to bind RNA polymerase and transcriptioninitiation sequences for ribosome binding. For example, a bacterialexpression vector includes a promoter such as the lac promoter and fortranscription initiation the Shine-Dalgarno sequence and the start codonAUG (Sambrook et al., Molecular Cloning, A Laboratory Manual 2d Ed.(Cold Spring Harbor, N.Y., 1989), or Ausubel et al., Current Protocolsin Molecular Biology (Greene Assoc., Wiley Interscience, NY, N.Y.,1992). Similarly, a eukaryotic expression vector includes a heterologousor homologous promoter for RNA polymerase II, a downstreampolyadenylation signal, the start codon AUG, and a termination codon fordetachment of the ribosome. Such vectors can be obtained commercially orassembled by the sequences described in methods well known in the art,for example, the methods described above for constructing vectors ingeneral. Expression vectors are useful to produce cells that express theinvention hybrid/chimeric transactivator (fusion) polypeptide.

This invention provides a transformed host cell that recombinantlyexpresses the invention hybrid/chimeric transactivator (fusion)polypeptides. Invention host cells have been transformed withrecombinant nucleic acid molecules encoding chimeric transactivatorscomprising a DNA binding domain of a mammalian or non-mammalianhypoxia-inducible factor protein and a functional transcriptionalactivator domain of a transcriptional activator protein. An example is amammalian cell comprising a plasmid adapted for expression in amammalian cell. The plasmid contains a polynucleotide encoding a DNAbinding domain of a mammalian or non-mammalian hypoxia-inducible factorprotein and a functional transcriptional activator domain of atranscriptional activator protein and the regulatory elements necessaryfor expression of the invention hybrid/chimeric transactivator (fusion)polypeptides.

Appropriate host cells include bacteria, archebacteria, fungi,especially yeast, plant cells, insect cells and animal cells, especiallymammalian cells. Of particular interest are E. coli, B. subtilis,Saccharomyces cerevisiae, SF9 cells, C129 cells, 293 cells, Neurospora,and CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloidand lymphoid cell lines. Preferred replication systems include M13,ColE1, SV40, baculovirus, lambda, adenovirus, artificial chromosomes,and the like. A large number of transcription initiation and terminationregulatory regions have been isolated and shown to be effective in thetranscription and translation of heterologous proteins in the varioushosts. Examples of these regions, methods of isolation, manner ofmanipulation, and the like, are known in the art. Under appropriateexpression conditions, host cells can be used as a source ofrecombinantly produced invention hybrid/climeric transactivator (fusion)protein.

Nucleic acids (polynucleotides) encoding invention hybrid/chimerictransactivator (fusion) polypeptides may also be incorporated into thegenome of recipient cells by recombination events. Otherrecombination-based methods such as nonhomologous recombinations ordeletion of endogenous gene by homologous recombination, especially inpluripotent cells, may also be used.

Targeting invention vectors to target or host cells may be accomplishedby linking a targeting molecule to the vector. A targeting molecule isany agent that is specific for a cell or tissue type of interest,including for example, a ligand, antibody, sugar, receptor, or otherbinding molecule. The ability of targeted vectors renders inventionvectors particularly useful in the treatment of hypoxia-associateddisorders.

Transfer of the polynucleotide/transgene to the target or host cells byinvention vectors can be evaluated by measuring the level of thepolynucleotide/transgene product in the target or host cell. The levelof polynucleotide/transgene product in the target or host cell directlycorrelates with the efficiency of transfer of thepolynucleotide/transgene by invention vectors.

Expression of the polynucleotide/transgene can be monitored by a varietyof methods known in the art including, inter alia, immunological,histochemical and activity assays. Immunological procedures useful forin vitro detection of the hybrid/chimeric transactivator (fusion)polypeptide in a sample include immunoassays that employ a detectableantibody. Such immunoassays include, for example, ELISA, Pandexmicrofluorimetric assay, agglutination assays, flow cytometry, serumdiagnostic assays and immunohistochemical staining procedures which arewell known in the art. An antibody can be made detectable by variousmeans well known in the art. For example, a detectable marker can bedirectly or indirectly attached to the antibody. Useful markers include,for example, radionuclides, enzymes, fluorogens, chromogens andchemiluminescent labels.

For in vivo imaging methods, a detectable antibody can be administeredto a subject, tissue or cell and the binding of the antibody to thepolynucleotide/transgene product can be detected by imaging techniqueswell known in the art. Suitable imaging agents are known and include,for example, gamma-emitting radionuclides such as ¹¹¹In, ^(99 m)Tc, ⁵¹Crand the like, as well as paramagnetic metal ions, which are described inU.S. Pat. No. 4,647,447. The radionuclides permit the imaging of tissuesby gamma scintillation photometry, positron emission tomography, singlephoton emission computed tomography and gamma camera whole body imaging,while paramagnetic metal ions permit visualization by magnetic resonanceimaging.

The present invention provides isolated hybrid/chimeric transactivator(fusion) peptide(s), polypeptide(s) and/or protein(s) encoded by theinvention nucleic acids. As used herein, the term “isolated” means aprotein molecule free of cellular components and/or contaminantsnormally associated with a native in vivo environment. Inventionpolypeptides and/or proteins include any naturally occurring allelicvariant, as well as recombinant forms thereof. Invention polypeptidescan be isolated using various methods well known to a person of skill inthe art.

The methods available for the isolation and purification of inventionfusion proteins include, precipitation, gel filtration, andchromatographic methods including molecular sieve, ion-exchange, andaffinity chromatography using e.g. HIF-1α-, EPAS1-, or HLF-specificantibodies or ligands. Other well-known methods are described inDeutscher et al., Guide to Protein Purification: Methods in EnzymologyVol. 182, (Academic Press, 1990). When the invention polypeptide to bepurified is produced in a recombinant system, the recombinant expressionvector may comprise additional sequences that encode additionalamino-terminal or carboxy-terminal amino acids; these extra amino acidsact as “tags” for immunoaffinity purification using immobilizedantibodies or for affinity purification using immobilized ligands.

An example of the means for preparing the invention hybrid/chimerictransactivator (fusion) polypeptide(s) is to express inventionpolynucleotides in a suitable host cell, such as a bacterial cell, ayeast cell, an amphibian cell (i.e., oocyte), an insect cell (i.e.,drosophila) or a mammalian cell, using methods well known in the art,and recovering the expressed polypeptide, again using well-knownmethods. Invention polypeptides can be isolated directly from cells thathave been transformed with expression vectors, described herein in moredetail. The invention hybrid/chimeric transactivator (fusion)polypeptide, biologically active fragments, and functional equivalentsthereof can also be produced by chemical synthesis. As used herein,“biologically active fragment” refers to any portion of the polypeptidethat can assemble into an active protein. Synthetic polypeptides can beproduced using Applied Biosystems, Inc. Model 430A or 431A automaticpeptide synthesizer (Foster City, Calif.) employing the chemistryprovided by the manufacturer.

Modification of the invention nucleic acids, polynucleotides,polypeptides, peptides or proteins with the following phrases:“recombinantly expressed/produced”, “isolated”, or “substantially pure”,encompasses nucleic acids, polynucleotides, polypeptides, peptides orproteins that have been produced in such form by the hand of man, andare thus separated from their native in vivo cellular environment. As aresult of this human intervention, the recombinant nucleic acids,polynucleotides, polypeptides, peptides and proteins of the inventionare useful in ways that the corresponding naturally occurring moleculesare not, such as identification of selective drugs or compounds.

The present invention provides for non-human transgenic animals carryingtransgenes encoding chimeric transactivator proteins. These transgenicanimals may further comprise a gene of interest under the control ofhypoxia responsive elements (HREs). In various embodiments of theinvention, the transactivator protein may constitutively enhance theexpression of the gene of interest. Alternatively, the transactivatorprotein may only enhance the expression of the gene of interest undercertain conditions; for example, and not by way of limitation, byinduction. The recombinant DNA molecules of the invention may beintroduced into the genome of non-human animals using any method forgenerating transgenic animals known in the art.

The invention provides a transgenic non-human mammal that is capable ofexpressing nucleic acids encoding invention hybrid/chimerictransactivator (fusion) polypeptides.

Also provided is a transgenic non-human mammal capable of expressingnucleic acids encoding invention hybrid/chimeric transactivator (fusion)polypeptides so mutated as to be incapable of normal activity.

The present invention also provides a transgenic non-human mammal havinga genome comprising antisense nucleic acids complementary to nucleicacids encoding invention hybrid/chimeric transactivator (fusion)polypeptides so placed as to be transcribed into antisense mRNAcomplementary to mRNA encoding invention fusion polypeptides, whichhybridizes thereto and, thereby, reduces the translation thereof. Thepolynucleotide may additionally comprise an inducible promoter and/ortissue specific regulatory elements, so that expression can be induced,or restricted to specific cell types. Examples of non-human transgenicmammals are transgenic cows, sheep, goats, pigs, rabbits, rats and mice.Examples of tissue specificity-determining elements are themetallothionein promoter and the T7 promoter.

Animal model systems which elucidate the physiological and behavioralroles of invention polypeptides are produced by creating transgenicanimals in which the expression of the polypeptide is altered using avariety of techniques. Examples of such techniques include the insertionof normal or mutant versions of nucleic acids encoding invention fusionpolypeptides by microinjection, retroviral infection or other means wellknown to those skilled in the art, into appropriate fertilized embryosto produce a transgenic animal. See, for example, Carver, et al.,Bio/Technology 11:1263–1270, 1993; Carver et al., Cytotechnology9:77–84, 1992; Clark et al, Bio/Technology 7:487–492, 1989; Simons etal., Bio/Technology 6:179–183, 1988; Swanson et al., Bio/Technology10:557–559, 1992; Velander et al., Proc. Natl. Acad. Sci., USA89:12003–12007, 1992; Hammer et al., Nature 315:680–683, 1985;Krimpenfort et al., Bio/Technology 9:844–847, 1991; Ebert et al.,Bio/Technology 9:835–838, 1991; Simons et al., Nature 328:530–532, 1987;Pittius et al., Proc. Natl. Acad. Sci., USA 85:5874–5878, 1988;Greenberg et al., Proc. Natl. Acad. Sci., USA 88:8327–8331, 1991;Whitelaw et al., Transg. Res. 1:3–13, 1991; Gordon et al.,Bio/Technology 5:1183–1187, 1987; Grosveld et al., Cell 51:975–985,1987; Brinster et al., Proc. Natl. Acad. Sci., USA 88:478–482, 1991;Brinster et al., Proc. Natl. Acad. Sci., USA 85:836–840, 1988; Brinsteret al., Proc. Natl. Acad. Sci., USA 82:4438–4442, 1985; Al-Shawi et al.,Mol. Cell. Biol. 10(3):1192–1198, 1990; Van Der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148–6152, 1985; Thompson et al., Cell56:313–321, 1989; Gordon et al., Science 214:1244–1246, 1981; and Hoganet al., Manipulating the Mouse Embryo: A Laboratory Manual (Cold SpringHarbor Laboratory, 1986).

Another technique, homologous recombination of mutant or normal versionsof these genes with the native gene locus in transgenic animals, may beused to alter the regulation of expression or the stricture of theinvention polypeptides (see, Capecchi et al., Science 244:1288, (1989);Zimmer et al., Nature 338:150, (1989)). Homologous recombinationtechniques are well known in the art. Homologous recombination replacesthe native (endogenous) gene with a recombinant or mutated gene toproduce an animal that cannot express native (endogenous) protein butcan express, for example, a mutated protein which results in alteredexpression of invention fusion polypeptides.

In contrast to homologous recombination, microinjection adds genes tothe host genome, without removing host genes. Microinjection can producea transgenic animal that is capable of expressing both endogenous andexogenous polypeptides. Inducible promoters can be linked to the codingregion of the nucleic acids to provide a means to regulate expression ofthe transgene. Tissue-specific regulatory elements can be linked to thecoding region to permit tissue-specific expression of the transgene.Transgenic animal model systems are useful for in vivo screening ofcompounds for identification of ligands, i.e., agonists and antagonists,which activate or inhibit polypeptide responses.

This invention further provides a composition containing an acceptablecarrier and any of an isolated, purified hybrid/chimeric transactivator(fusion) polypeptide, an active fragment thereof, or a purified, matureprotein and active fragments thereof, alone or in combination with eachother. These polypeptides or proteins can be recombinantly derived,chemically synthesized or purified. As used herein, the term “acceptablecarrier” encompasses any of the standard pharmaceutical carriers, suchas phosphate buffered saline solution, water and emulsions such as anoil/water or water/oil emulsion, and various types of wetting agents.

As used herein the term “effective amount” refers to an amount thatalleviates the deficiency by the sustained production of biologicallyactive chimeric human-viral transactivator protein in the cells of anindividual. Sustained production of biologically active chimerichuman-viral transactivator protein in individuals can be evaluated bythe alleviation of the symptoms associated with hypoxia-associateddisorders, for example ischemic heart disease, peripheral vasculardisease, ischemic limb disease, and the like. The precise effectiveamount of vector to be used in the method of the present invention canbe determined by one of ordinary skill in the art with consideration of,for example, individual differences in age, weight, extent of diseaseand condition of the individual.

The present invention further provides a method for providingbiologically active chimeric human-viral transactivator protein to thecells of an individual with a hypoxia-associated disorder comprisingintroducing into a such individual an amount of invention vectorseffective to infect and sustain expression of biologically activechimeric human-viral transactivator protein in cells in which theassociated transcription factor is absent, insufficient or nonfunctionaltherein. Invention vectors may be delivered to the target cells as apharmaceutical composition comprising the vector and a pharmaceuticallyacceptable carrier. The vector may be delivered to target cells bymethods known in the art, for example, intravenous, intramuscular,intranasal, subcutaneous, intubation, lavage, and the like.

Accordingly, the present invention provides alternative approaches inwhich the expression of a range of potentially beneficial genes isinduced following the expression of biologically active mammaliantranscription factors.

HIF-1α was cloned by PCR from HeLa cell cDNA and inserted into theexpression vector, pcDNA3 (Clontech, Palo Alto, Calif.; Invitrogen, SanDiego, Calif.). In this plasmid, expression of the gene is controlled bythe CMV promoter. Following confirmation of the structure of theconstruct by sequencing, it was tested in HeLa and 293 cells bycotransfection with reporter plasmids in which transcription of theluciferase gene is controlled by either the EPO promoter/enhancer or theVEGF promoter. Induction of HIF-1α activity was accomplished bytreatment of the cells with wither CoCl₂ or desferrioxamine, both ofwhich are known to induce HIF-1α activity by a mechanism similar tohypoxia. This assay confirmed that the HIF-1α protein was indeed active.

Two HIF-1α/VP16 hybrids were constructed, the first was truncated atamino acid 390 of HIF-1α, the second at amino acid 530. Thetransactivation domain of HSV VP16 (aa413–490) was then joineddownstream. These proteins were tested by cotransfection into HeLa and293 cells with either the EPO-luciferase or VEGF-luciferase reporterplasmids as described above. The results show that in both cell types,the levels of luciferase gene expression observed in cells cotransfectedwith the hybrids is higher (as much as 20–100×) than that obtained withHIF-1α even when exposed to induction by CoCl₂ or desferrioxamine. Thisresult implies that the hybrid proteins are indeed active and that theVP16 domain confers strong transactivation activity even in the absenceof induction (i.e., under normoxic conditions). This suggests that thesehybrid/chimeric fusion proteins might be a viable therapeutic forischemic heart disease.

The present invention is further illustrated by the following exampleswhich in no way should be construed as being further limiting. Thecontents of all references cited throughout this application are herebyexpressly incorporated by reference.

EXAMPLES Example 1 Hybrid/Chimera Construction

A hybrid transcription factor (pcDNA3/HIF.VP-16.Af12) composed of a DNAbinding and dimerization domains from HIF-1α and the transactivationdomain from herpes simplex virus VP16 (FIG. 1) was constructed toprovide strong, constitutive activation of genes normally involved inthe physiological adaptation to hypoxia. As is described below, weanalyzed the effect of this HIF-1α/VP16 transcription factor on VEGFgene expression in vitro, and on neovascularization in a rabbit hindlimbischemia model.

Recombinant Plasmids

The full-length (aa1–826) HIF-1α gene was isolated by PCR (AdvantagecDNA PCR Kit, Clontech, Palo Alto, Calif.) from a HeLa cell cDNA library(Clontech) using the primers set forth in SEQ ID NO's 1 and 2 andinserted between the KpnI and XbaI sites of the expression vector,pcDNA3 (Invitrogen, Carlsbad, Calif.). In this plasmid, gene expressionis controlled by the cytomegalovirus (CMV) immediate earlyenhancer/promoter. The HIF-1α/VP-16 hybrid was constructed by truncatingHIF-1α at aa390 (an Af12 site) and then joining the transactivationdomain of HSV VP-16 downstream. A VP16 fragment (aa 413–490) with Af12and XbaI ends was amplified by PCR using Vent polymerase (New EnglandBiolabs, Beverly, Mass.) and the primers set forth in SEQ ID NO's 3 and4 and this fragment was cloned into the appropriate sites of thepcDNA3/HIF-1α construct. A related construct (pcDNA3/HIF/VP-16/R1) wasproduced by truncating HIF-1α at aa530 by partial digestion with EcoR1(FIG. 2). The integrity of all sequences generated by PCR was verifiedby DNA sequencing using an Applied Biosystems 377 DNA Sequencer. Allcloning manipulations were carried out following standard procedures(Sambrook, J. et al., Molecular Cloning, A Laboratory Manual 2d Ed.(Cold Spring Harbor, N.Y., 1989)). Restriction enzymes and DNA-modifyingenzymes were obtained from ether New England Biolabs or LifeTechnologies, Inc. (Gaithersburg, Md.)and used according to themaunfacturer's specifications. Plasmid DNAs were purified with kitsobtained from Qiagen (Chatsworth, Calif.). The plasmid constructexpressing human VEGF₁₆₅ (phVEGF₁₆₅) has been described previously(Tsurumi, et al., Circulation 96:II-382–II-388 (1997)). Luciferasereporter plasmids (EPO-luc and VEGF-luc) were generously provided by Dr.H. Franklin Bunn (Brigham and Women's Hospital, Harvard Medical School).

Transient Transfections

HeLa cells were grown in Dulbecco's modified Eagles medium-high glucose(DME; Irvine Scientific, Santa Ana, Calif.) supplemented with 10% fetalbovine serum (FBS; JRH Biosciences, Lenexa, Kans.). For the luciferasereporter experiments, HeLa cells (3×10⁵ cells/60 mM dish) weretransfected using the calcium phosphate ProFection kit (Promega,Madison, Wis.) according to manufacturer's instructions. Duplicatedishes were transfected with 5 μg of each plasmid (HIF-1α construct andeither the EPO-luc (Blanchard, K. L., et al. Mol. Cell. Biol. 12:5373–85(1992)) or the VEGF-luc (Levy, A. P., et al., J. Biol. Chem.270:13333–40 (1995)) reporter as well as pCMVβ (Clontech). At 24 hrpost-transfection, one set of dishes was induced with 100 μMdesferrioxamine. The cells were harvested 18 hr post-induction andluciferase activity was assayed (Promega). β-galactosidase activity ineach sample was also determined using the Galacto-Light kit (Tropix,Bedford, Mass.) and these values were used to normalize the luciferaseactivity as a control for transfection efficiency.

Example 2 Activity of HIF-1α and HIF-1α/VP16 Chimeric/Hybrid Constructsin vitro

The HIF-1α/VP16 hybrid constructs were tested for their ability toactivate gene expression by cotransfection along with reporter plasmidsinto either 293 or HeLa cells (FIGS. 3 and 4, respectively). Thereporter plasmids used in these experiments contained the luciferasegene under the transcriptional control of either the erythropoietinenhancer/promoter (Blanchard, K. L., et al., Mol. Cell. Biol. 12:5373–85(1992)) or the VEGF promoter (Levy, A. P. et al., J. Biol. Chem.270:13333–40 (1995)). (In FIGS. 3–5, pcDNA3/HIF/VP16.Af12 andpcDNA3/HIF/VP16.R1 are referred to as “HIF-4/VP-16.Af1II” and“HIF-4/VP-16.R1”, respectively.)

Prior to transfection, cells were plated in 6 cm dishes at a density of1×10⁶ (239) or 2.5×10⁵ (HeLa) cells per dish. Cells were co-transfectedwith the HIF-1α or HIF-1α/VP16 expression plasmids and a luciferasereporter plasmid containing either the EPO enhancer/promoter or the VEGFpromoter.

Transfection was carried out by calcium phosphate precipitation usingthe ProFection™ kit (Promega, Madison, Wis.) using 5μg each of theHIF-1α/VP16 expressing constructs and the reporter plasmid per dish. At24 hours post-transfection, cells were induced by adding fresh mediacontaining either 100 μg/ml CoCl₂ or 100 μg/ml desferrioxamine. At 16hours post-induction, the cells were harvested and the luciferaseactivity was measured using a luciferase assay kit (Promega) accordingto the manufacturer's instructions.

Induction of HIF-1 activity was simulated by treatment withdesferrioxamine, an iron chelator, which acts via a mechanism similar tohypoxia. As was predicted for the HIF-1α/VP-16 hybrid transcriptionfactor, luciferase expression was constitutive and not dependent ofdesferrioxamine induction. Luciferase expression in uninduced anddesferrioxamine-treated cells was not significantly different. Incontrast, luciferase activity in cells transfected with the full-lengthHIF-1α construct was increased 4–5 fold following treatment withdesferrioxamine. Furthermore, transcriptional activation achieved by theHIF-1α/VP16 hybrid in uninduced cells was 10–20-fold higher than thatobserved in desferrioxamine-treated cells transfected with thefull-length HIFα. Overall, levels of luciferase expression activated byHIF-1α were not much higher than the background levels attributable toendogenous HIF-1 activity in the HeLa cells. As shown in FIG. 6,subsequent studies have confirmed these findings. FIG. 6, left, showsluciferase activity produced using an EPO 3′enhancer-promoter-luciferase reporter construct. FIG. 6, right, showsluciferase activity produced using a VEGF promoter-luciferase reporterconstruct.

Example 3 VEGF Production in Response to the HIF-1α/VP16 HybridConstructs

To determine if the HIF-1α/VP16 hybrids were capable of activatingexpression of an endogenous VEGF gene, these constructs were transfectedinto HeLa cells and VEGF was assayed by ELISA. Prior to transfection,HeLa cells were plated in 6 cm dishes at a density of 2.5×10⁵ cells perdish. The cells were transfected with 10 μg of the HIF-1α/VP16 hybridplasmid DNA. At 24 hours post-transfection, cells in duplicate plateswere either control (not-induced) or induced by adding fresh mediacontaining 100 μg/ml desferrioxamine. At 40 hours post-induction, themedia was harvested and assayed for VEGF by ELISA, (Quantikine™, R&DSystems, Minneapolis, Minn.) according to the manufacturer'sinstructions.

The results from this assay (FIG. 5) show that the hybridHIF-1α/VP16/Af1II construct (truncated at amino acid 390 of HIF-1α) wasable to activate expression of VEGF even in the absence of induction.The concentration of VEGF in the media from theHIF-1α/VP16/Af1II-transfected cells, however, was not higher than thatobserved in mock-transfected or HIF-1α-transfected cells with induction.This suggests that perhaps a maximum plateau level is achieved by theendogenous HIF-1α present in HeLa cells. The important observation,however, is that this level can be achieved with the HIF-1α/VP16/Af1IIhybrid in the absence of induction.

The same result was not achieved with the second hybrid construct,HIF-1α/VP16/R1 (truncated at amino acid 530 of HIF-1α), although thisconstruct had been active in the luciferase reporter assay. Additionalin vitro analyses have confirmed an apparent difference in activitybetween the two constructs. Specifically, the HIF-1α/VP16/Af12 construct(truncated at amino acid 390 of HIF-1α) is more active than the longerHIF-1α/VP16/R1 construct, both with respect to activation oftranscription of luciferase reporter constructs and up-regulation ofendogenous VEGF and EPO gene expression in HeLa and Hep3B cells. Thisdifference in activity may be due to the presence in the longerconstruct of a portion of an “oxygen-dependent domain” of HIF-1α whichis reported to confer instability under normoxic conditions, (see Huanget al., PNAS 95:7987–7992 (1998)).

In additional analyses of VEGF production, HeLa cells (3×10⁵ cells/60 mMdish) were transfected with Lipofectamine (Life Technologies, Inc; 3.7μg DNA, 14 μl of Lipofectamine) in Opti-MEM media (Life Technologies,Inc.) for 17 hr. Seven hours later, one set of dishes was treated with100 μM desferrioxamine. At 42 hr post-induction, the culture medium washarvested and the cells were lysed in 250 μl lysis buffer (0.5% NP-40, 1mM EDTA, 50 mM Tris (pH 8.0), 120 mM NaCl, 100 μM PMSF, 0.1 U/mlAprotinin, 1 μM Pefabloc, 5 μg/ml Leupeptin). VEGF concentration wasassayed as above and the total cell protein was analyzed using theBio-Rad (Hercules, Calif.) protein assay. ELISA values were normalizedto total cell protein.

Rat C6 glioma cells (American Type Culture Collection, Rockville Md.)were seeded onto 12-well culture plates 3 days before transfection andcultured with DME (Life Technologies, Inc) supplemented with 10% FBS(Sigma, St. Louis, Mo.). Transfection was performed essentially asdescribed previously (Lee, E. R. et al. Hum. Gene Ther. 7:1701–1717(1996)). Briefly, plasmid DNA was complexed with an equal volume ofcationic lipid GL#67 to obtain a final concentration of 80:20 μM(DNA:lipid). Lipid/DNA complexes (400 μl/well, approximately 5×10⁵cells/well) were added to the cells in Opti-MEM media and incubated for5 hrs. The complexes were then removed and cells returned to DME+10%FBS. The medium was replaced with fresh medium (1 ml/well) with orwithout desferrioxamine (100 μM) 24 hr after transfection. Twenty-fourhours later, the medium was collected for analysis of VEGF productionand the cells were lysed in 500 μl cell lysis buffer. VEGF productionwas assayed using an ELISA kit specific for mouse VEGF (R&D Systems) andvalues were normalized to total cell protein.

In both HeLa cells and C6 cells the HIF-1α/VP16 hybrid constructsignificantly enhanced production of VEGF in the absence ofdesferrioxamine treatment. In HeLa cells, VEGF production inHIF-1α/VP16-transfected cells was further enhanced by desferrioxamine,but in C6 cells, no difference was observed. Background levels of VEGFexpression were much higher in HeLa than in the C6 cells. A much greatereffect of the HIF-1α/VP16 hybrid on the magnitude of VEGF expression wasobserved in the C6 cells (5-fold greater than untransfected orHIF-1α-transfected cells treated with desfcrrioxamine). In HeLa cells,VEGF production in uninduced HIF-1α/VP16-transfected cells wascomparable to that in untransfected cells exposed to desferrioxamine.However, in both cell lines, the absolute amount of VEGF (2.3 pg/μgprotein) produced in uninduced, HIF-1α/VP16-transfected cells was almostidentical. These results suggest that the desferrioxamine-induced levelof endogenous HIF-1α protein and/or HIF-1α activity is greater in HeLathan in C6 cells; this may be explained by a species or celltype-specific difference. Notably, the HIF-1α/VP16 hybrid, based on ahuman transcription factor, is able to activate the rat VEGF gene,suggesting conservation of HREs across species. Sequence analysis of the5′-flanking region of the rat VEGF gene has revealed several potentialHREs; the position of one of these is conserved between the rat andhuman genes. This element is located within a 28 bp region that isidentical in the rat and human VEGF genes.

Example 4 Analysis of the HIF-1α/VP16 Hybrid Transcription Factor in theRabbit Hindlimb Ischemia Model

A. Serum VEGF Level

Naked plasmid DNA encoding either the HIF-1α/VP16 hybrid gene(pHIF-1α/VP16) or human VEGF₁₆₅ (phVEGF₁₆₅) was administered byinjection into the medial large, adductor and semimembranous muscles ofrabbits in which the femoral artery had been excised to induce hindlimbischemia. Serum VEGF levels were assayed by an ELISA assay preparedagainst human VEGF. Accordingly, this assay as applied to theHIF-1α/VP16-treated group may not be quantitative as the endogenousrabbit protein is produced in these animals. However, the kinetics andpersistance of VEGF expression may be compared among the groups. Beforetreatment serum VEGF levels were almost undetectable and similar foreach group. However, at 3 days post-administration, the VEGF levelsincreased to 10.5 (±3.9) pg/ml in the phVEGF₁₆₅-transfected animals andto 26.3 (±4.6) pg/ml in the pHIF-1α/VP16-treated group. The VEGF levelsin the pHIF-1α/VP16-treated animals were still high, yet reduced(14.9±3.0 pg/ml) at 5 days after treatment. In contrast, at 5 days,serum VEGF was undetectable in the animals treated with phVEGF₁₆₅. VEGFwas not detected in the control (pCMVβ-treated) group either before orat 3 and 5 days after treatment. Though VEGF protein levels haddecreased by five days, expression of both transgenes, analyzed byRT-PCR, persisted to 14 days post-administration.

B. Red Blood Cell Measurement

The red blood cell count, hematocrit and hemoglobin values beforetreatment (day 10) and 30 days after treatment (day 40) for thephVEGF₁₆₅, pHIF-1α/VP16, and control groups are shown in Table 1 below.There was no difference in red blood cell count and hemoglobin among thethree groups either before or after treatment. Although the hematocritappeared to increase in the pHIF-1α/VP16-treated animals, a similarincrease was observed in the control group, suggesting that this resultwas not due to expression of the HIF-1α/VP16 hybrid transcriptionfactor.

C. Lower-limb Calf Blood Pressure

At 10 days after induction of ischemia (immediately before plasmidinjection), calf blood pressure ratio (ischemic/normal limb) was similaramong the three groups (phVEGF₁₆₅=0.45±0.01, pHIF-1α/VP16=0.44±0.02,control=0.45±0.03; P=NS). By day 40 (30 days after transfection), theblood pressure ratio had improved among the three groups. The bloodpressure ratio in the pHIF-1α/VP16-treated animals (0.93±0.02), however,was significantly higher (P<0.01) than in those that received phVEGF₁₆₅(0.82±0.03)or the control group (0.69±0.02). The blood pressure ratio atday 40 was higher (P<0.01) in the phVEGF₁₆₅-treated group than in thecontrols.

D. Intravascular Doppler Guide Wire Measurements

Resting blood flow and maximal blood flow (papaverine-stimulated) in theischemic limb were similar among the three groups at day 10. However, atday 40, the resting and papaverine-stimulated maximal blood flow in thepHIF-1α/VP16-transfected (41.6±3.1 mL/min and 111.2±5.7 mL/min,respectively) and phVEGF₁₆₅-transfected groups (42.2±3.9 mL/min and88.7±7.4 mL/min, respectively) were significantly higher than that ofthe control group (28.7±1.5 mL/min and 65.3±3.8 mL/min, respectively).The resting flow was similar between pHIF-1α/VP16 and phVEGF₁₆₅-treatedrabbits at day 40; however, the maximal flow was significantly higher(p<0.05) in the animals transfected with pHIF-1α/VP16 than in thephVEGF₁₆₅-treated group. Resting and maximal blood flow in thenonischemic limb were similar among the 3 groups at day 10 as well asday 40.

E. Effect of HIF-1α/VP16 and VEGF₁₆₅ on Collateral Vessel Development

Quantitative analysis of angiographically visible collateral vessels onthe medial thigh was performed by determining vascular density. Atbaseline (day 10) before treatment, there was no significant differencein angiographic score among the phVEGF₁₆₅, pHIF-1α/VP16 and controlgroups (0.38±0.03, 0.42±0.01, 0.41±0.02, respectively; P=NS). By day 40,the angiographic scores in the pHIF-1α/VP16-treated (0.61±0.01) and inthe phVEGF₁₆₅-treated (0.58±0.03) rabbits were significantly higher thanthat of the control group (0.51±0.05). There was no statisticallysignificant difference in angiographic score at 40 days betweenpHIF-1α/VP16-treated and phVEGF₁₆₅-treated groups. The principal findingaccounting for the increase in angiographic score observed in theanimals that received pHIF-1α/VP16 and phVEGF₁₆₅ was enhancement inso-called mid-zone collateral vessels.

To further evaluate the effect of intramuscular HIF-1α/VP16 and VEGFgene therapy upon revascularization of the ischemic hindlimb, the medialthigh muscles of the ischemic limbs were histologically examined at day40. Capillary densities observed in the muscles of thepHIF-1α/VP16-treated group (255±13/mm²) and phVEGF₁₆₅-treated group(210±10/mm²) were significantly higher than that of the control group(150±4/mm²). In addition, the capillary density was higher (p<0.05) inthe animals transfected with pHIFα/VP16 than in the phVEGF₁₆₅-treatedanimals. Moreover, the capillary/muscle fiber ratios of the pHIF-1α/VP16and phVEGF₁₆₅-transfected rabbits (0.88±0.06 and 0.75±0.03,respectively) were significantly higher than the control (0.58±0.03;results not shown). Light microscopic signs of frank myonecrosis werenot observed in either group.

Clinical Implications

Activation of VEGF gene expression by the HIF-1α/VP16 hybrid factor invitro and in vivo suggests that the VP16 activation domain is able tointeract with the accessory factors required for expression of the HIF-1target gene in the cell types examined (human cervical epithelia, ratglioma, rabbit skeletal muscle). Thus, administration of the HIF-1α/VP16hybrid via gene therapy may prove to be an effective treatment forischemia associated with vascular disease. In this application,HIF-1α/VP16 may up-regulate a variety of genes, including VEGF.Therapeutic benefit may be achieved, not only as a result of stimulationof angiogenesis, but also through additional HIF-1-mediated localadaptations to low oxygen tension such as vasodilation and up-regulationof anaerobic metabolism. The administration of growth factors and/or thegenes encoding them to enhance angiogenesis may be useful as a treatmentfor tissue ischemia in cases where conventional revascularizationtechniques are not feasible.

The enhanced potency observed with the HIF-1α/VP16 hybrid relative toVEGF₁₆₅ in vivo might be attributed to several factors. First, thelevels of expression of VEGF promoted by HIF-1α/VP16 appreared to exceedthat achieved with the VEGF₁₆₅ plasmid. Moreover, VEGF expressionappeared to persist longer in the pHIF-1α/VP16-treated animals than inthose that received phVEGF₁₆₅, since at 5 days post-administration,serum VEGF was detected in the former group but not in the latter.

F. Procedures

Intramuscular Gene Transfer

Twenty-nine rabbits were used to study the effect of intramuscular genetherapy on hindlimb ischemia. All protocols were approved by St.Elizabeth's Institutional Animal Care and Use Committee. Male New ZelandWhite rabbits (4.0 to 4.3 kg) (Pine Acre Rabbitry, Norton, Mass.) wereanesthetized with a mixture of ketamine (50 mg/kg) and acepromazine (0.8mg/kg) after premedication with xylazine (2 mg/kg). The surgicalprocedures have been previously described (Takeshita, S. et al., Am. J.Physiol. 93:662–670 (1994a)). Briefly, the femoral artery was completelyexcised from its proximal origin as a branch of the external iliacartery to the point distally where it bifurcates into the saphenous andpopliteal arteries. All animals were closely monitored followingsurgery. Analgesia (0.25 mg/kg levophanol tartrate; Hoffmann-La RocheInc., Nutley, N.J.) was administered subcutaneously for one day.Prophylactic antibiotics (enrofloxacin, Bayer Corporation, ShawneeMission, Kans.) were also administered subcutaneously for a total of 5days postoperatively.

An interval of 10 days was allowed for postoperative recovery, afterwhich the rabbits were returned to the catheterization laboratory.Following completion of baseline measurements, four different sites inthree major thigh muscles received direct injections with plasmid DNA orvehicle only (normal saline) with the use of a 3-ml syringe and a25-gauge needle advanced through a small skin incision. For eachinjection, the tip of the needle was inserted typically to a depth of 3to 5 mm in the medial large (two sites), adductor (one site), andsemimembranous (one site) muscle. The detailed procedure for the muscleinjection has been previously described (Tsurumi et al., Circulation94:3281–3290 (1996)). This technique was used to administer 500 μg ofpHIF-1α/VP16 (n=11), 500 μg of phVEGF₁₆₅ (n=10), or 500 μg of pCMVβ(n=8). One hundred and twenty five μg in 0.5 ml of normal saline wasinjected at each of four sites for a total of 500 μg/2.0 ml for eachanimal. After the completion of four injections, the skin was closedwith 4-0 nylon sutures.

Immunoassay of Serum VEGF

For the first 6 rabbits in each group, blood samples were drawn from thecentral artery of the rabbit ear using a 23-gauge needle immediatelybefore treatment as well as 3 and 5 days after plamid transfection. Theblood sample was stored at 4° C. for 30 minutes and then centrifuged at3,000 rpm for 15 minutes. Serum was frozen at −80° C. until assay ofVEGF by ELISA (R&D Systems). The lower limit of detection of serum VEGFwas 9.0 pg/mL. The assay was performed in duplicate for each sample. Theintra-observer coefficient of variation was less than 8%.

Red Blood Cell, Hematocrit and Hemoglobin Measurement

Blood samples were drawn from the central artery of the rabbit car usinga 23-gauge needle immediately before treatment (day 10) and at the dayof sacrifice (day 40). The red blood cell, hematocrit and hemoglobinwere measured by a commercially automatic detector (Sysmex alpha, SysmexCorporation, Long Grove, Ill.).

Calf Blood Pressure Ratio

Calf blood pressure was measured in both hindlimbs using a Dopplerflowmeter (Model 1050; Parks Medical Electronics, Aloha, Oreg.)immediately before treatment (day 10) as well as one month afterinitiation of the therapy (day 40). On each occasion, with rabbits underanesthesia, the hindlimbs were shaved and cleaned. The pulse of theposterior tibial artery was identified using a Doppler probe and thesystolic blood pressure in both limbs was measured using standardtechniques (Takeshita, S. et al., Am. J. Physiol. 93:662–670 (1994a)).The calf blood pressure ratio was defined for each rabbit as the ratioof systolic pressure of the ischemic limb to that of the normal limb.

In Vivo Doppler Flow Measurement

Blood flow was quantified in vivo before selective internal iliacangiography on days 10 and 40 with a 0.018-in Doppler guide wire(Cardiometrics, Inc., Mountain View, Calif.) as previously described(Banters, C. et al., Am J Physiol. 267:H1263–H1271 (1994)). The wire tipwas positioned at the origin of the common iliac artery to the proximalsegment of the internal iliac artery supplying the ischemic limb. Timeaverage of the spectral peak velocity (APV) was recorded at rest andmaximal APV was recorded after bolus injection of 2 mg of papaverine(Sigma, St. Louis, Mo.).

Doppler-derived blood flow (Q_(D)) was calculated asQ_(D)=(πd²/4)(0.5×APV), where d is vessel diameter, and APV is timeaverage of the spectral peak velocity. The luminal diameter of the iliacartery was determined angiographically with an automated edge-detectionsystem that has been validated previously in vivo. The vascular diameterwas measured at the site of the Doppler sample volume (5 mm distal tothe wire tip). Cross-sectional area was calculated assuming a circularlumen. The mean velocity was estimated as 0.5×APV by assuming atime-averaged parabolic velocity profile across the vessel. TheDoppler-derived flow calculated in this fashion has been shown tocorrelate with flow measurements determined by electromagneticflowmeters both in vitro and in vivo (Tsurumi et al., Circulation94:3281–3290 (1996)). Because 2 mg of papaverine had no effect on vesseldiameter (Ku, D. D., et al., Am. J. Physiol. 265:H586–H592 (1993)), thediameter measurements were used to calculate both rest and maximum flow.

Selective Angiography

Selective internal iliac angiography was performed as previouslydescribed (Takeshita, S. et al. Am. J. Physiol. 93:662–670 (1994a)). Thetip of the catheter was positioned in the internal iliac artery at thelevel of interspace between the seventh lumbar and the first sacralvertebrae. A total of 5 ml of nonionic contrast media (Isovue-370;Squibb Diagnostics, New Brunswick, N.J.) was injected with an automatedangiographic injector at a flow rate of 1 ml/s through a 3F infustioncatheter (Tracker-18; Target Therapeutics, Fremont, Calif.) positionedin the internal iliac artery. Serial images of the ischemic hindlimbwere then recorded on 105-mm spot film at a rate of one film per secondfor at least 10 seconds. After completion of angiography, the catheterwas removed and the wound was closed.

Quantitative angiographic analysis of collateral vessel development wasperformed using a grid overlay comprised of 2.5-mm-diameter circlesarranged in rows spaced 5 mm apart. This overlay was placed over themedial thigh area of the 4-s angiogram. The total number of gridintersections in the medial thigh area, as well as the total number ofintersections crossed by a contrast-opacified artery, were counted in asingle blind fashion. An angiographic score was calculated for each filmas the ratio of grid intersections crossed by opacified arteries dividedby the total number of grid intersections in the medial thigh.

Capillary Density

Vascular density was evaluated at the microvascular level using lightmicroscopic sections taken from the ischemic hindlimbs. Tissue specimenswere -obtained as transverse sections from the adductor muscle andsemimembranous muscle of the ischemic limb when the animals weresacrificed. Muscle samples were embedded in OCT compound (Miles,Elkhart, Ind.), snap-frozen in liquid nitrogen, and cut into 5-um-thicksections. Tissue sections were stained for alkaline phosphatase with anindoxyl-tetrazolium method to detect capillary endothelial cells aspreviously described (Ziada, A. M., et al., Cardiovasc. Res. 18:724–732(1984)) and then were counterstained with eosin. A total of 20 differentfields from the two muscles were randomly selected, and the number ofcapillaries and myofibers counted under a 20× objective. The capillarydensity (capillaries/mm²) and the capillary-to-myocyte ration were thendetermined.

Example 5 HIF-1α/VP16 Recombinant Adenoviruses

As an alternative to naked DNA, adenoviral vectors were constructedcarrying the HIF-1α-based genetic construct, as described below.

Two HIF-1α/VP16 hybrids were cloned into a previral plasmid vector (CMVpromotor, SV40 pA, p1X-; called “empty vector” (EV)—obtained originallyfrom R. Doll). As is described above, these two hybrid proteins containthe DNA-binding and dimerization domains from HIF-1α (the first istruncated at aa390 of HIF-1α at an AF111 site; the second is truncatedat aa 530 at EcoR1 site) and the transactivation domain from HerpesSimplex Virus VP-16 protein.

Shuttle vector EV/HIF-1α/VP-16.Af111 was linearized with BstB1 andshuttle vector EV/HIF-1α/VP16.R1 was linearized with BamH1. 15 μg of DNAof each vector was digested for 4 hours, then purified twice byphenol/chloroform extraction and ethanol precipitation. Pellets wereresuspended in 50 μl of 0.1×TE and used for transfection (2 μl of eachdigested DNA were run on a gel prior to transfection).

Preparation of Viral Backbone DNA

5 μg of Ad2CMVBgal-6 DNA were digested with PshAI and SnaBI at 37° C.for about 24 hours, and 1/10 of the reaction was run on 0.8% Agarose/TBEgel to check the completion of digestion. The rest of the reaction wasused for transfection.

The Ad2CMVBgal-6 backbone is the same as that of the vector described inthe Example of U.S. Pat. No. 5,707,618, incorporated herein by referencein its entirety, except that it contains the B-gal gene inserted intothe deleted E1 portion of the vector. Specifically, this vector isdeleted for Ad E1, and E4 sequences are deleted from the ClaI site at34077 to the TaqI site at 35597. The ORF6 sequence from 33178 to 34082is inserted into the E4 region. The SV40 early polyA sequence isinserted adjacent to the ORF6, which also serves to prevent readthroughfrom the ORF6 gene into the L5 (fiber) sequences. Protein IX isrepositioned from its original location in the virus genome into theE4-deleted region as a BamH1 fragment. The protein IX fragment containsits own promoter, and may be cloned into the vector in either direction.The construct is shown in FIG. 3 of the '618 patent.

Transfection

Transfection was conducted using the standard protocol from Promega's“Profection-Calcium Phosphate Transfection Kit,” as follows.

Day One: Plate 293 cells on 60 mm dish at 1×10⁶/dish and incubate at 37°C./5% CO₂ for 24 hours.

Day Two:

-   1. Four hours prior to the transfection, remove the medium from the    cells and replace it with 4.5 ml of fresh growth medium.-   2. Prepare the DNA and CaCl₂ mixture:

 4 μg of viral backbone DNA (Ad2CMVBgal-6) 120 μl 10 μg of shuttlevector (EV + Afl11,EV + RI)  48 μl  2 M CaCl₂  37 μl ddH₂O  95 μlin a 1.5 ml eppendorf tube and mix gentle. Leave mixture at roomtemperature for 15–30 minutes.

-   3. Precipitate DNA-CaCl₂ with 300 μl of 2×HBS and incubate at RT for    0.5 to 2 hours. Then add precipitated DNA to cells and incubate for    16–17 hours.    Day Three:-   Change Medium.    Day Five:-   Split cells from 60 mm dish to 100 mm dish.    Day Seven:-   Split cells from 100 mm dish to 150 mm dish.    Day Nine:-   Pick up 5 plagues of each transfection.    Day Eleven:-   Pick up 12 more plagues from EV+RI and 10 more plagues from    EV+Af111. The plagues in 250 μl of growth medium were frozen and    thawed 3 times and stored at −20° C.    Initial Screenings of Recombinant Adenoviruses-   1. Infection    Day One:-   Plate cells on 24 well plates at 1.4×10⁵ cells/well.    Day Two:-   Infect cells (about 40–50% confluent) with 200 μl of each plague at    37° C./5% CO₂ for 2 hours and then add 800 μl of growth medium.    Replace cells in incubator and grow until CPE occurs.    Day Four:-   Harvest one well of each plate when CPE is advanced.    Day Five:-   Wash off CPE cells from 10 wells of EV+Af111 and 13 wells of EV+RI.    Day Eight:-   Wash off remaining CPE cells.-   Freeze/thaw CPE cell lysate 3 times.-   2. Preparation of Viral DNA from CPE Cell Lysate

250 μl of CPE

250 μl of 2× proteinase K lysis buffer

40 μl of 10 mg/ml proteinase K

Cells were incubated at 37° C. for 3 hours and extracted 2 times withphenol/chloroform. Samples were precipitated with ethanol at 20° C. o/Nand pellets were resuspended in 25 μl of ddH₂O. (Lysis buffer: 40 mMEDTA; 40 Mm Tris-HCl, PH 8; 2% SDS)

-   3. Viral DNA Digestion    25 μl of DNA was digested with restriction enzyme BcII at 50° C. O/N    and then separated on 0.8% agarose/TBE gel. All of the plagues were    true recombinants. There was no background. A schematic of the    adenoviral sequences contained within the HIF-1α/VP16 adenoviral    vectors is shown in FIG. 7. This vector is identical to the    Ad2CMVBgal-6 backbone except that the B-gal gene has been replaced    with the HIF-1α/VP16 hybrid construct.

Example 6 HIF-1α/NFκB Hybrid Transactivators

Using techniques similar to those described in Example 1, we constructeda nucleic acid sequence encoding a chimeric transactivator proteincomprising a DNA binding and dimerization domain from HIF-1α and atransactivation domain from NF-κB. Specifically, a DNA fragment codingfor the-activation domain of the p65 subunit of NFκB (Schmitz, M. L. andBaeuerle, P. A., 1991 EMBO J. 10:3805–3817, Schmitz, M. L. et al., 1994J. Biol. Chem. 269:25613–25620, Schmitz, M. L. et al., 1995, J. Biol.Chem. 270:15576–15584) was generated by PCR amplification of the DNAsequence (Advantage cDNA PCR kit, Clontech) from a HeLa cell cDNAlibrary (Clontech). The DNA fragment was then inserted between the Af12and XbaI sites of the pcDNA3/HIF-1α expression vector. This construct(pHIF-1α/NF-κB) therefore consists of aa 1–390 of HIF-1α and aa 407–551of the NF-κB p65 subunit. The integrity of sequences generated by PCRwas confirmed by DNA sequencing.

Initial in vitro experiments were performed to evaluate the ability ofthe HIF-1α/NF-κB hybrid to activate expression of the endogenous VEGFgene in HeLa cells. The pHIF-1α/NF-κB construct was transfected intoduplicate plates of HeLa cells in parallel with the pHIF-1α/VP16construct and pHIF-1α (full-length, wild-type HIF-1α gene) as a control,using methods similar to those described in Example 3. Twenty-four hoursafter transfection, one set of plates were exposed to desferrioxaminefor induction of HIF-1α activity, the other set was left untreated. Themedia was harvested 48 hr after induction for assay of VEGF by ELISA (R& D Systems). As shown in FIG. 8 top, the pHIF-1α/NF-κB constructappears to be constitutively active, i.e. the level of VEGF detected inuninduced cells was relatively high, equal to that of induced,mock-transfected cells. However, the overall level of VEGF expression incells transfected with the pHIF-1α/NF-κB construct was not as high asthat observed in cells transfected with pHIF-1α/VP16.

A similar experiment was performed to evaluate up-regulation oferythropoietin (EPO) gene expression by pHIF-1α/NF-κB followingtransfection into Hep3B cells. As with VEGF, transfection of the cellswith pHIF-1α/NF-κB results in expression of endogenous EPO in theabsence of induction, however the levels of EPO are lower than thoseobserved in cells transfected with pHIF-1α/VP16 (FIG. 8, bottom).

Although the invention has been described with reference to thedisclosed embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the claimswhich follow the References.

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1. A nucleic acid molecule encoding a biologically active chimerictransactivator protein comprising: (a) the DNA binding domain of a humanhypoxia inducible factor protein; and (b) a protein domain capable oftranscriptional activation which is not derived from a hypoxia induciblefactor protein.
 2. The nucleic acid molecule according to claim 1,wherein said protein domain capable of transcriptional activation isderived from a protein selected from the group consisting of: HSV VP16,NEκB, a heat shock factor; p53; fos; v-jun; factor EF-C; HIV tat; HPVE2; Ad E1A; Sp1; AP1; CTF/NF1; E2F1; HAP1; HAP2; MCM1; PHO2; GAL4, GCN4,and GAL11.
 3. The nucleic acid molecule according to claim 2, whereinsaid protein domain capable of transcriptional activation is atranscriptional activation domain from HSV VP16.
 4. The nucleic acidmolecule according to claim 2, wherein said protein domain capable oftranscriptional activation is a transcriptional activation domain fromNFκB.
 5. An expression vector comprising the nucleic acid moleculeaccording to any one of claims 1–4, operatively linked to an expressioncontrol sequence.
 6. The expression vector according to claim 5, whereinsaid expression control sequence comprises an inducible promoter.
 7. Theexpression vector according to claim 5, wherein said vector is anadenoviral vector.
 8. The expression vector according to claim 7,wherein said expression control sequence comprises an induciblepromoter.
 9. An isolated host cell comprising the expression vectoraccording to claim
 5. 10. An isolated host cell comprising theexpression vector according to claim
 6. 11. An isolated host cellcomprising the expression vector according claim
 7. 12. An isolated hostcell comprising the expression vector according claim
 8. 13. Apharmaceutical composition comprising the expression vector according toclaim 5 and a pharmaceutically acceptable carrier.
 14. A pharmaceuticalcomposition comprising the expression vector according to claim 6 and apharmaceutically acceptable carrier.
 15. A pharmaceutical compositioncomprising the expression vector according to claim 7 and apharmaceutically acceptable carrier.
 16. A pharmaceutical compositioncomprising the expression vector according to claim 8 and apharmaceutically acceptable carrier.
 17. A method for increasing theexpression of a hypoxia-inducible gene in a mammalian cell, said methodcomprising the step of introducing into said mammalian cell in vitro theexpression vector according to claim 5 so as to increase the expressionof said hypoxia inducible gene in said cell.
 18. A method for increasingthe expression of a hypoxia-inducible gene in a mammalian cell, saidmethod comprising the step of introducing into said mammalian cell invitro the expression vector according to claim 6 so as to increase theexpression of said hypoxia inducible gene in said cell.
 19. A method forincreasing the expression of a hypoxia-inducible gene in a mammaliancell, said method comprising the step of introducing into said mammaliancell in vitro the expression vector according to claim 7 so as toincrease the expression of said hypoxia inducible gene in said cell. 20.A method for increasing the expression of a hypoxia-inducible gene in amammalian cell, said method comprising the step of introducing into saidmammalian cell in vitro the expression vector according to claim 8 so asto increase the expression of said hypoxia inducible gene in said cell.21. A method for reducing ischemic tissue damage in a mammalian subjecthaving a hypoxia-associated disorder comprising the step ofadministering at the site of said ischemic tissue damage in said subjectan effective amount of the pharmaceutical composition according to claim13.
 22. A method for reducing ischemic tissue damage in a mammaliansubject having a hypoxia-associated disorder comprising the step ofadministering at the site of said ischemic tissue damage in said subjectan effective amount of the pharmaceutical composition according to claim14.
 23. A method for reducing ischemic tissue damage in a mammaliansubject having a hypoxia-associated disorder comprising the step ofadministering at the site of said ischemic tissue damage in said subjectan effective amount of the pharmaceutical composition according to claim15.
 24. A method for reducing ischemic tissue damage in a mammaliansubject having a hypoxia-associated disorder comprising the step ofadministering at the site of said ischemic tissue damage in said subjectan effective amount of the pharmaceutical composition according to claim16.